Neuromics

Lineage Selection-Neural Stem Cells for SC Grafts

2012-01-26T16:21:00.000-08:00

Our Neural Progenitor Markers keep moving up in the "hit parade". These markers are important for lineage selection. This selection is essential to circumvent the possibility of tumor formation and facilitate the safe translation of ES-based therapies to humans.

Here's a recent pub referencing use of several of our markers for selecting or confirming lineage: J. Simon Lunn, Crystal Pacut, Emily Stern, Stacey A. Sakowski, J. Matthew Velkey, Sue O'Shea, Eva Feldman. Intraspinal transplantation of neurogenin-expressing stem cells generates spinal cord neural progenitors. dx.doi.org/10.1016/j.nbd.2011.12.044...Tuj1 (Neuromics, 1:1000), Nestin (Neuromics, 1:500)...
Highlights: Expression of appropriate transcription factors is one approach to direct the differentiation of ES cells towards a specific lineage and stop proliferation. Neural differentiation can be initiated in ES cells by expression of Neurogenin1 (Ngn1). In this study we investigate the effects of controlled Ngn1 expression on mouse ES (mES) cell differentiation in vitro and following grafting into the rat spinal cord. In vitro, Ngn1 expression in mES cells leads to rapid and specific neural differentiation, and a concurrent decrease in proliferation. Similarly transplantation of Ngn1-expressing mES cells into the spinal cord lead to in situ differentiation and spinal precursor formation. These data demonstrate that Ngn1 expression in mES cells is sufficient promote neural differentiation and inhibit proliferation, thus establishing an approach to safely graft ES cells into the spinal cord.

Image: Neural progenitors were labeled with anti-rat Nestin polyclonal antibody (Cat#:GT15114) and stained with conjugated donkey anti-goat secondary anti-body (green). Differentiated neurons were labeled with neuron-specific mouse anti-β-III tubulin/ Tuj1-(Cat#MO15013 monoclonal antibody (red). Nuclei were stained with DAPI (blue).

I will continue to post updates on the application of Neuromics' Stem Cell Markers

Early Diagnosis of Diabetic Retinopathy

2012-01-22T16:32:00.000-08:00

The earlier the diagnosis the better the outcome. This is especially true with autoimmune diseases like Diabetic Retinopathy (DR). DR is the leading cause of blindness among persons of working age in the industrialized world. Here I feature a publication that shows axoglial alterations at the distal portion of the optic nerve could be the first structural change in the diabetic visual pathway. This could prove good news for discovering better therapies thus preventing blindness: Diego C. Fernandez, Laura A. Pasquini, Damián Dorfman, Hernán J. Aldana Marcos, Ruth E. Rosenstein. Early Distal Axonopathy of the Visual Pathway in Experimental Diabetes. doi:10.1016/j.ajpath.2011.09.018

Oligodendrocytes are responsible for insulating axons. Disruptions in the formation of oligodendrocytes could initiate the domino effect that leads to decreasing and eventual total loss of vision. The authors, for example, discovered that in diabetic rats, oligodendrocyte lineage (OL) cells showed hypertrophic somas and a high number of processes.

Images/Data: OL linage evaluation. Immature OL (O1+ cells) and OL precursor (PDGFR-α+ cells) were evaluated by immunostaining and measured as optical density (OD) per section. In the distal ON from animals that were diabetic for 6 weeks, significantly increased O1 and PDGFR-α immunostaining was observed, with the presence of disorganized and hypertrophic cells. Data are mean ± SEM (n = 5 animals per group); *P < 0.01 versus age-matched controls, by Student′s t-test. Scale bar = 50 μm.

At the ultrastructural level, alterations and loss of larger axons were observed in the distal ON from animals that were diabetic for 6 weeks. In these fibers, myelin was highly disorganized, and frequent lamellar membranous bodies were observed.

I will track new develops in this research and post relevant results here.

Primary Neuron Assays for Studying Neurodegeneration

2012-01-16T10:31:00.000-08:00

Our goal is to provide our customers and collaborators the tools they need to insure success. This is defined by having the specific Primary Neurons, Growth Factor plus the Markers to meet unique research needs.

The proof is in the results. Here are some highlights.

Images/Data: FIGURE 5. Microglial p38α MAPK-dependent TNFα is involved in LPS-induced neurite degeneration. (A) Photomicrographs of MAP-2 immunocytochemistry show the morphology of neurons after 72h of co-culture with microglia. The arrow points to the appearance of neurites that have been damaged by LPS-activated WT microglia. In contrast, the arrowhead points to the morphological appearance of healthy, undamaged neurites. (B) Diagram of the Sholl method for quantifying the total number of healthy neurites that intersect the concentric circles. (C) Quantification of healthy neurites by the Sholl analysis demonstrates that LPS stimulation of p38α WT microglia in co-culture causes neurite degeneration as seen by a significant reduction in the number of intersections by healthy neurites in the LPS-stimulated group compared to the unstimulated group (white bars). This degeneration can be attenuated by the addition of a blocking antibody to TNFα (5μg/ml), while the non-immune IgG control was not protective (gray bars). Microglia from p38α KO mice stimulated with LPS (black bar) also have significantly less neurite degeneration than the LPS-stimulated p38α WT microglia (white bar). However, by adding TNFα back to the p38α KO microglia co-culture, there is a significant decrease in the healthy neurite arborization compared to the p38α KO microglia stimulated with LPS alone (black bars). (***p<0.005; Bonferroni’s multiple comparison test). Data represents 2 independent experiments. Scale bar equals 25μm. Molecular Neurodegeneration 2011, 6:84 doi:10.1186/1750-1326-6-84

hN2 cells grown in culture for 4 days and stained with our chicken polyclonal to Neurofilament light or low molecular weight chain NF-L, a marker of neurons. Many of the differentiating cells show strong cytoplasmic and clearly fibrillar staining for NF-L. Blue stain is DAPI and reveals cell nuclei of some non neuronal cells in this culture.

We will continue to post relevant images and data that demonstrate our capabilities.

TRPV1s in Action

2012-01-13T07:21:00.000-08:00

Our TRPV1s continue to be widely used and published. This recent publication features use out TRPV1-C guinea pig polyclonal for immunohistochemistry and TRPV1-mouse specific for Western Blotting: Sarah E. Canetta, Edlira Luca, Elyse Pertot, Lorna W. Role, David A. Talmage. Type III Nrg1 Back Signaling Enhances Functional TRPV1 along Sensory Axons Contributing to Basal and Inflammatory Thermal Pain Sensation. PLoS ONE 6(9): e25108. doi:10.1371/journal.pone.0025108...IHC: TRPV1 (guinea pig, 1:1000, GP14100 Neuromics); WB: TRPV1 (rabbit, 1:1000, RA14113 Neuromics).

Figure. Sensory axons, but not soma, from Type III Nrg1+/− mice show reduced capsaicin responsiveness compared to axons from WT mice. (A) Representative traces of intracellular calcium along sensory axons in response to 1 µM capsaicin or 56 mM KCl. The change in intracellular calcium from baseline over time ([(F−F0)/F0]*100) is shown for WT (left) and Type III Nrg1+/− (right) axons. Hatched diagonal lines indicate where the time course was non-continuous. (B) Quantification of the maximum change in intracellular calcium in response to application of 1 µM capsaicin or 56 mM KCl by genotype. Averages of 5 animals per genotype were compared using a Student's t-test. Type III Nrg1+/− axons showed a significantly decreased response to capsaicin (p<0.05), but not to KCl, relative to WTs. Graph shows mean±SEM. (C) Type III Nrg1+/− sensory soma show normal response to capsaicin. Quantification of maximal change in fluorescence from baseline ([(F−F0)/F0]*100) in WT or Type III Nrg1+/− sensory neuron soma in response to 1 µM capsaicin or 56 mM KCl. Average responses from 4 WT and 4 Type III Nrg1+/− animals to application of capsaicin or KCl were compared by genotype using a Student's t-test. There was no statistically significant difference between genotypes. Graphs show mean±SEM. (D) Type III Nrg1 (green) and TRPV1 (red) are co-expressed along P21 WT cultured sensory neuron axons identified with a pan-axonal (PA) marker (blue). White arrows indicate examples where Type III Nrg1 and TRPV1 are in close proximity. Scale bar equals 10 µm. (E) P21 WT and Type III Nrg1+/− sensory neuron cultures have equivalent levels of total TRPV1 protein. Total TRPV1 protein measurement by immunoblot. The 95 kD TRPV1 band and the 35 kD GAPDH band are shown from a representative experiment comparing protein from P21 WT and Type III Nrg1+/− cultures. Quantification of fold change in intensity of TRPV1:GAPDH normalized to WT average. There was no statistically significant change in the ratio of TRPV1 to GAPDH between genotypes (WT, Type III Nrg1+/−, n = 3 animals). Genotype comparisons were made using a Student's t-test. Graph shows mean±SEM. doi:10.1371/journal.pone.0025108.g004
All TRPV1 Publications.

Primary Neurons vs PC12 cells for Compound Testing

2012-01-07T08:37:00.000-08:00

This publication compares PC12 Cells vs E18 Primary Cortical Neurons. The cells showed permeability to some key compounds where the Neurons did not. This demonstrates the importance of including primary neurons in compound testing assays for Neuro-disease research: Wei Zhang , Radhia Benmohamed, Anthony C. Arvanites, Richard I. Morimoto, Robert J. Ferrante, Donald R. Kirsch, Richard B. Silverman. Cyclohexane 1,3-diones and their inhibition of mutant SOD1-dependent protein aggregation and toxicity in PC12 cells. Bioorganic & Medicinal Chemistry. Elsevier Ltd. All rights reserved.doi:10.1016/j.bmc.2011.11.039.
Abstract: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the progressive loss of motor neurons. Currently, there is only one FDA-approved treatment for ALS (riluzole), and that drug only extends life, on average, by 2–3 months. Mutations in Cu/Zn superoxide dismutase (SOD1) are found in familial forms of the disease and have played an important role in the study of ALS pathophysiology. On the basis of their activity in a PC12-G93A-YFP high-throughput screening assay, several bioactive compounds have been identiﬁed and classiﬁed as cyclohexane-1,3-dione (CHD) derivatives. A concise and efﬁcient synthetic route has been developed to provide diverse CHD analogs. The structural modiﬁcation of the CHD scaffold led to the discovery of a more potent analog (26) with an EC50 of 700 nM having good pharmacokinetic properties, such as high solubility, low human and mouse metabolic potential, and relatively good plasma stability. It was also found to efﬁciently penetrate the blood–brain barrier. However, compound 26 did not exhibit any signiﬁcant life span extension in the ALS mouse model. It was found that, although 26 was active in PC12 cells, it had poor activity in other cell types, including primary cortical neurons, indicating that it can penetrate into the brain, but is not active in neuronal cell potentially due to poor selective cell penetration. Further structural modiﬁcation of the CHD scaffold was aimed at improving global cell activity as well as maintaining potency. Two new analogs (71 and 73)
were synthesized, which had signiﬁcantly enhanced cortical neuronal cell permeability, as well as similar
potency to that of 26 in the PC12-G93A assay. These CHD analogs are being investigated further as novel
therapeutic candidates for ALS.

see: Bioorg. Med. Chem. 2011, 19, 613. and J. Med. Chem. 2012, in press

Related Links: Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes.

Image: E18 hippocampal neurons stained with Tau (red) and Doublecortin (green). The two proteins overlap in the proximal dendrites (yellow) Axons (low doublecortin content) are red. Blue staining is the nuclear DNA.

Salivary Neuropeptides Y2s and Satiety

2011-12-23T10:50:00.000-08:00

A possible new slant for combating obesity

The researchers in this study acheived a sustained increased PYY_3-36 (a neuropeptide) expression via viral vector-mediated gene delivery targeting salivary glands and the good news: this increase resulted in a significant long-term reduction in food intake (FI) and body weight (BW). This is evidence for new functions of the previously characterized gut peptide PYY_3-36 suggesting a potential simple and efficient alternative therapeutic approach for the treatment of obesity: Andres Acosta1, Maria D. Hurtado1, Oleg Gorbatyuk, Michael La Sala, David Duncan, George Aslanidi, Martha Campbell-Thompson, Lei Zhang, Herbert Herzog, Antonis Voutetakis, Bruce J. Baum, Sergei Zolotukhin. Salivary PYY: A Putative Bypass to Satiety. PLoS ONE 6(10): e26137. doi:10.1371/journal.pone.0026137. Received: July 22, 2011; Accepted: September 20, 2011; Published: October 10, 2011...Rabbit anti-Y2R (Dilution: 1:3000) using TSA...

Images: A) Immunolocalization of Y2R-positive cells in the hippocampus of C57Bl/6J mouse (WT), a (+) control. (B) Immunolocalization of Y2R in the tongue epithelia of Y2R KO mouse, a (-) control. VEG – von Ebner's gland. (C) Immunolocalization of Y2R-positive cells in the CV area of the tongue of a C57Bl/6J mouse. (D) close-up of (C). (E), and (F) close ups of (D), top and bottom rectangles, respectively. doi:10.1371/journal.pone.0026137.g003

These findings could prove a first step in identifying new targets for obesity fighting therapies. Increasing PYY salivary output would provide satiety with less food intake. This would give a whole new perspective on dieting. If we are satsified with less food intake is this really dieting? Stay tuned.

Opioid Addiction During Pregnancy-Implications for Neuro-development

2011-12-03T07:18:00.000-08:00

I would like to thank Dr. Carmen Sato-Bigbee, Virginia Commonwealth University School of Medicine, for kindly sharing this important study. The publication also references use of our Mu Opioid and Nociceptin/Orphanin FQ Receptor Antibodies: Andrew C. Eschenroeder, Allison A. Vestal-Laborde, Emilse S. Sanchez, Susan E. Robinson, Carmen Sato-Bigbee. Oligodendrocyte responses to buprenorphine uncover novel and opposing roles of μ-opioid- and nociceptin/orphanin FQ receptors in cell development: Implications for drug addiction treatment during pregnancy. Glia Volume 60, Issue 1, pages 125–136, January 2012.

Highlights: Oligodendrocytes are responsible for making myelin in the CNS. The authors have shown We have shown previously that rat brain myelination is significantly altered by buprenorphine, an opioid analogue currently used in clinical trials for managing pregnant opioid addicts. In this study, perinatal exposure to low levels of this drug induced accelerated and increased expression of myelin basic proteins (MBPs), cellular and myelin components that are markers of mature oligodendrocytes. In contrast, supra-therapeutic drug doses delayed MBP brain expression and resulted in a decreased number of myelinated axons. We have now found that this biphasic-dose response to buprenorphine can be attributed to the participation of both the l-opioid receptor (MOR) and the nociceptin/orphanin FQ receptor (NOP receptor) in the oligodendrocytes. This is the first study showing the potential role of the NOP receptor in myelination.

High levels of opiate exposure could negatively disrupt the normal interplay between these two systems altering the developmental pattern of brain myelination. Understanding this pathway, could help researchers find ways to favorably modulate myelination and protect neuro-development of fetuses exposed to high levels of opiates during pregnancy.

Related Data:

Direct treatment of immature oligodendrocytes with buprenorphine alters MBP expression in a dose-specific manner. Cells isolated from 9-day-old rat brains were incubated for 4 days in CDM with or without 0.25, 0.5, 1.0, and 3.0 lM buprenorphine. MBP levels were determined by western blotting using b-actin levels as loading controls. Figures correspond to representative experiments. Results in the bar graph are expressed as percentage of controls (0 lM buprenorphine) 6 SEM from five experiments and correspond to the combined scanning of the four major MBP isoforms. **P <0.005 and ***P<0.0001.

Pre-oligodendrocytes express both MOR and the NOP receptor. Cells isolated from 9-day-old rat brain were allowed to fully attachon the culture plates by overnight incubation and stained by double
immunocytochemistry with O4 (green) together with anti-MOR or anti-NOP receptor antibodies (red). Scale bar: 20 lm. The western blot shows MOR and NOP receptor expression in two different samples of developing oligodendrocytes directly isolated from 9-day-old rat brains.

I will be posting future studies investigating the molecular mechanisms by which buprenorphine and methadone affect myelination and neuron-glial interactions. These should provide deeper understanding into these developmental processes and new and better strategies for the managing of both pregnant addicts and drug addiction in adolescence.

IF Staining of Human Primary Neurons

2011-11-25T06:13:00.000-08:00

Primary Neurons are inputs or raw materials for cell based assays. When cells do not work as promised, there are multiple costs including lost time and potentially flawed data. Neuromics strives to provide easy to culture, potent and cost effective cells. Proving these capabilities is an ongoing activity for us. This includes testing these cells using our markers.

I wanted to share new immunofluorescence images. Here is a link to the protocol: staining primary neurons.

hN2 cells stained with our chicken polyclonal antibody to Vimentin, in red. Islands of Hn2 cells form after 4 days in culture forming beautiful flower like structures. Vimentin is a well established marker of early differentiating neuronal lineage cells. Taken with a 10X objective lens. Blue staining is the nuclear DNA.

hN2 cells grown in culture for 4 days and stained with our chicken polyclonal to MAP2, a marker of neurons. Differentiating cells show strong cytoplasmic staining for MAP2 . Blue stain is DAPI and reveals cell nuclei of some non neuronal cells in this culture.

Fear Changes Hippocampus Neuropns

2011-11-20T08:46:00.000-08:00

Fear in mice catalyzes rapid accumalation of EphrinB2 pyramidal neurons of the CA1 area.

This study suggests that rapid accumulation of EphrinB2 in hippocampal CA1 neurons is involved in the behavioural and cellular modifications induced by contextual fear conditioning. A similar mechanism does not appear to occur in lateral amygdala neurons, in spite of the robust behavioural and cellular modifications induced in such structure by cued fear conditioning: Antonio Trabalzaa, Sandra Colazingaria, Carmelo Sgobiob, Arturo Bevilacqua. Contextual learning increases dendrite complexity and EphrinB2 levels in hippocampal mouse neurons. Behavioural Brain Research. doi:10.1016/j.bbr.2011.11.008.

Diabetic retinopathy blindness-root causes

2011-11-11T08:48:00.000-08:00

Diabetic retinopathy is a leading cause of acquired blindness. This publication from our friends at University of Buenos Aires touches on potential root causes: Diego C. Fernandez, Laura A. Pasquini, Damián Dorfman, Hernán J. Aldana Marcos, Ruth E. Rosenstein. Early Distal Axonopathy of the Visual Pathway in Experimental Diabetes. doi:10.1016/j.ajpath.2011.09.018
" In animals that had been diabetic for 6 weeks, a large increase in astrocyte reactivity occurred in the distal (but not the intraorbital) portion, which coincided with significant axon loss. Moreover, profound myelin alterations and altered morphologic features of oligodendrocyte lineage were observed at the distal (but not the proximal) optic nerve portion. The present results suggest that axoglial alterations at the distal portion of the optic nerve could be the first structural change in the diabetic visual pathway."
The authors used our PDGFR Alpha/CD140A Marker to Study the change in Oligodendrocyte Lineage precursor cells. Expression of the protein was increased in these cells with the presence of disorganized and hypertrophic cells. This could disrupt formation of myelin resulting the pathological alteration at the distal portion.

Opioid Induced Itch

2011-11-08T11:08:00.000-08:00

Our widely used and frequently published Opioid Receptors Antibodies are used for a spectrum of pain research. Here is an interesting study on the root causes of opioid induced itch.

Xian-Yu Liu, Zhong-Chun Liu, Yan-Gang Sun, Michael Ross1, Seungil Kim, Feng-Fang Tsai, Qi-Fang Li, Joseph Jeffry, Ji-Young Kim, Horace H. Loh, Zhou-Feng Chen. Unidirectional Cross-Activation of GRPR by MOR1D Uncouples Itch and Analgesia Induced by Opioids. Cell, Volume 147, Issue 2, 14 October 2011, Pages 261-262.

Root Causes: Spinal opioid-induced itch, a prevalent side effect of pain management, has been proposed to result from pain inhibition. We now report that the μ-opioid receptor (MOR) isoform MOR1D is essential for morphine-induced scratching (MIS), whereas the isoform MOR1 is required only for morphine-induced analgesia (MIA). MOR1D heterodimerizes with gastrin-releasing peptide receptor (GRPR) in the spinal cord, relaying itch information. We show that morphine triggers internalization of both GRPR and MOR1D, whereas GRP specifically triggers GRPR internalization and morphine-independent scratching. Providing potential insight into opioid-induced itch prevention, we demonstrate that molecular and pharmacologic inhibition of PLCβ3 and IP3R3, downstream effectors of GRPR, specifically block MIS but not MIA. In addition, blocking MOR1D-GRPR association attenuates MIS but not MIA. Together, these data suggest that opioid-induced itch is an active process concomitant with but independent of opioid analgesia, occurring via the unidirectional cross-activation of GRPR signaling by MOR1D heterodimerization.

Mouse epiSCs Into Myelinating Cells

2011-10-26T10:38:00.000-07:00

This study published recently in Nature Methods hit my radar scope becaused it referenced use of our widely used and frequently published stem cell marker Tuj 1 (Neuron-specific class III beta-tubulin): Fadi J Najm, Anita Zaremba, Andrew V Caprariello, Shreya Nayak, Eric C Freundt, Peter C Scacheri, Robert H Miller & Paul J Tesar. Rapid and robust generation of functional oligodendrocyte progenitor cells from epiblast stem cells. Nature Methods (2011) doi:10.1038/nmeth.1712.

Dr. Paul Tesar and his team at Case Western University demonstrated the ability to convert pluripotent epiblast stem cells into pure populations of myelinating cells, called oligodendrocyte progenitor cells (OPCs). First, stem cells in a petri dish are treated with molecules to direct them to become the most primitive cells in the nervous system. To produce OPCs, these primitive cells are treated with a defined set of proteins. The cells were cultured on laminin and treated withh apporopriate growth factors. The OPCs were nearly homogenous and could be multiplied to obtain more than a trillion cells.

The OPCs were treated with thyroid hormone, which is key to regulating the transition of the OPCs to oligodendrocytes. The result was the OPCs stopped proliferating and turned into oligodendrocytes within four days.

These methods could used to potentially produce stable and pure populations of human OPCs in a significant enough number to treat patients with demyelinating diseases such as multiple sclerosis and cerebral palsy.

Immunostaining Neurons and Glia

2011-10-20T04:25:00.000-07:00

I would like to thank Dr. Gerry Shaw, University of Florida for his excellent work with our Primary Neurons and Astrocytes and Neuronal-Glial Markers. Here's an example image with many more to follow:

Image: E18 hippocampal neurons stained with MAPT (red) and Doublecortin (green). The two proteins overlap in the proximal dendrites, but doublecortin is more abundant in the growth cones and periphery. As a result, the periphery appears green while the more proximal regions of the cells are yellow. The single longer process of this cell, presumably an axon, has a low doublecortin content and so appears red. Blue staining is the nuclear DNA. Protocol on datasheet.

BDNF and Exercise Study-Running Mice

2011-10-09T18:01:00.000-07:00

Get fit and get smart. There is increasing evidence that vigorous exercise increases secretion of Brain-Derived Neurotrophic Factor (BDNF) Protein. BDNF is a catalyst of processes that increase growth of neurons especially in the hippocampus.

In this study researchers show new cell proliferation, survival, neuron number, and neurotrophin levels were enhanced only when running was accessible to mice. They conclude that exercise is the critical factor mediating increased BDNF levels and adult hippocampal neurogenesis: Tali Kobilo, Qing-Rong Liu, Kriti Gandhi, Mohammed Mughal, Yavin Shaham and Henriette van Praag. Running is the neurogenic and neurotrophic stimulus in environmental enrichment. doi: 10.1101/lm.2283011. Learn. Mem. 2011. 18: 605-609... human recombinant BDNF (0.1 µg) monomer (Neuromics)...
BDNF, CF Recombinant Protein
Related Reagents:
Neuron-Glial Expressed-Includes Neurotrophin Proteins
Neurotrophins and Growth Factor Antibodies
Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes.

Keep your brain healthy.

Immune-Inflammatory Response and Pain Research

2011-09-19T09:33:00.000-07:00

Our Pain and Inflammation Related Research Antibodies are increasingly being used to study the root causes of immune/inflammatory related pain induction. Here're related publications: Lintao Qu, Pu Zhang, Robert H. LaMotte, Chao Ma. Neuronal Fc-gamma receptor I mediated excitatory effects of IgG immune complex on rat dorsal root ganglion neurons. Brain, Behavior, and Immunity. Volume 25, Issue 7, October 2011, Pages 1399-1407......rabbit-anti-TRPV1, 1:1000, Neuromics...

Highlights: Pain often accompanies antigen-specific immune-related disorders though little is known of the underlying neural mechanisms. A common feature among these disorders is the elevated level of antigen-specific immunoglobulin (Ig) G in the serum and the presence of IgG immune complex (IC) in the affected tissue. We hypothesize that IC may directly activate the Fc-gamma receptor type I (FcγRI) expressed in nociceptive dorsal root ganglion (DRG) neurons and increase neuronal excitability thus potentially contributing to pain. Immunofluorescent labeling indicated that FcγRI, but not FcγRIIB or FcγRIII, was expressed in a subpopulation of rat DRG neurons including those expressing nociceptive markers. Calcium imaging revealed that the IC, but neither of the antibody (IgG) or antigen alone, produced an increase in intracellular calcium. This effect was abolished by the removal of the IgG Fc portion in the IC or the application of an anti-FcγRI antibody, suggesting a key role of the FcγRI receptor. Removal of extracellular calcium or depletion of intracellular calcium stores prevented the IC-induced calcium response. In whole-cell current-clamp recordings, IC depolarized the resting membrane potential, decreased the rheobase, and increased the number of action potentials evoked by a depolarizing current at 2× rheobase. In about half of the responsive neurons, IC evoked action potential discharges. These results suggest that a subpopulation of nociceptive neurons expresses functional FcγRI and that the activation of this receptor by IC increases neuronal excitability.

B. Huanga, X. Zhaoc, L.-B. Zhengb, L. Zhanga, B. Nia. Different expression of tissue inhibitor of metalloproteinase family members in rat dorsal root ganglia and their changes after peripheral nerve injury. Neuroscience, Volume 193, 13 October 2011, Pages 421-428....anti-P2X3 (rabbit, Neuromics, MN, USA)...

Xona Microfluidics and Neurons

2011-09-15T13:47:00.000-07:00

I am impressed with these Video from the Jeon Lab at UC Irvine. It represents a novel method for neuro-drug discovery:
Preparing E18 Cortical Rat Neurons for Compartmentalization in a Microfluidic Device.

This technology represents a way to separate axon from cell bodies.

hNP1™ Human Neural Progenitors Video Protocols

2011-09-03T09:50:00.000-07:00

Video Protocols designed to insure your success with our hNP1^TM Human Neural Progenitors

hNP1™ Human Neural Progenitors Video Protocols

Thawing, Culturing and Transfer-Sub Culturing Protocols.

Opioid Receptor Antibodies Trifecta

2011-08-30T05:44:00.000-07:00

This publication proposes a role for opioid receptors in treating cancers. It also references use of our μ, δ, and κ opioid receptor antibodies.

Kohei Yamamizu1, Sadayoshi Furuta, Shiori Katayama, Michiko Narita, Naoko Kuzumaki, Satoshi Imai, Hiroshi Nagase, Tsutomu Suzuki, Minoru Narita, and Jun K. Yamashita. The κ opioid system regulates endothelial cell differentiation and pathfinding in vascular development. Blood July 21, 2011 vol. 118 no. 3 775-785.

Highlights: The opioid system is, thus, a new regulator of vascular development that simultaneously modifies 2 distinct vascular properties, EC differentiation and vascular pathfinding. We confirmed that KOR, but not MOR, was highly expressed in various ECs such as HUVECs (data not shown), suggesting that KOR agonists could directly act on tumor ECs to suppress VEGF receptor expression, similar to the effects observed in embryonic ECs. If so, a combination therapy including an MOR agonist, morphine, and a KOR agonist (such as TRK820, a clinically approved drug in Japan for uremic pruritus) may prove useful for cancer therapy through the suppression of tumor angiogenesis by dual inhibition of VEGF ligands and receptors, extending the therapeutic benefits beyond pain relief.

Images: KOR was highly expressed in Flk1⁺ vascular progenitors. (A) RT-PCR showing mRNA expression of MOR, DOR, and KOR in ES cells, Flk1⁺ cells, cells after 1 or 3 days of Flk1⁺ cell culture (Flk-d1 or Flk-d3), CD31-positive cells (ECs) and CD31-negative cells (MCs) at Flk-d3. (B) Fluorescent staining for MOR, DOR, and KOR at Flk-d1. Nuclei are stained with DAPI (blue). Left, MOR; middle, DOR; right, KOR. Scale bars, 100 μm. (C) Double fluorescent staining for MOR, DOR, and KOR with CD31 (red) at Flk-d3. Nuclei are stained with DAPI (blue). Top, opioid (green) receptors (green); middle, CD31 (red); bottom, merged. Scale bars, 100 μm.

Is Neuropathy Really Gliopathy?

2011-08-27T09:20:00.000-07:00

I found this excellent website from posting by Dr. Jan M. Keppel Hesselink, Professor molecular pharmacology, director Institute neuropathic pain: http://www.neuropathie.nu/. It represents a new way of understanding root causes and potential therapies for Neuropathic Pain. Here're highlights:

Gliopathic pain: is a brand new term for what we always thought to be neuropathic pain. It refers to pain related to neuropathic pain, however, the primary driver of this pain is most probably more linked to glia and asterocytes. The mechanism of gliopathic pain is the hyperactivation of glia cells, which results in neuropathic pain.

The role of Glia and Astrocytes:Glia and astrocytes play a central role in neuropathic pain, and gliopathic pain, or asteropathic pain will become new synonyms for neuropathic pain. In a recent hallmark paper the term 'Gliopathic pain' was coined.

This is a reason to put our magnifying glass on glia. Gliamodulating drugs will become a new class of neuropathic drugs, the so called gliopathic modulating drugs, and the first prototype, the endogenous fatty acid palmitoylethanolamide, has already been explored in positive proof of principle studies.

For more than a century doctors are aware of the special properties of glia in response to injury. In Germany in 1894 professor Franz Nissl decribed the reaction of glial cells in relation to the nerve fibers in the spinal cord and highlighted their morphological changes after injury. Microglia becomes mores bigger and more abundant after injury and these glial responses can be seen as a biological reponse to promote nerve repair after injury. However, this response can go berzerk and might be one of the most important mechanisms leading to neuropathic pain.

This is a short synopsis. There is a wealth of more information on the website. That said, I will be posting more on Gliopathic Pain.

Primary Neurons and Cell Based Assays

2011-08-25T12:49:00.000-07:00

The feedback I receive from Neuroscientists is consistent. To paraphrase, "gives us healthy, consistent and potent primary cells. I understand the hard work it takes to generate meaningful and publishable results from cell based assays. Our Primary Neurons and Astrocytes are merely inputs for these assays. The real cost is the time invested in culturing and time lost if they don't work.

I have numerous postings on success: Primary Neurons Postings. I wanted to share more data and feedback.

Primary DRGs-Culturing these can be tricky. I make it a point to work with labs to make sure the protocol options best match the desired outcome for assays. This includes replacing cells to make sure we can accurately troubleshoot. This approach insures I can pin point the issues and make sure they are all resolved in round two. Here's a representative testimonial: "Thanks for following up, the DRGs worked great and we were able to get excellent data from them. Thanks so much for working with us." Adam Ross, Dr. Chengji Zhou Lab, UC Davis

Image: DRGs cultured on Calf Skin Collagen.

Primary Hippocampal Neurons-I would like to thank Vimal Swarup, University of Utah for this excellent image.

The cells have been fixed after 48 hrs, they were grown over poly-lysine coated coverslips in the media supplied by Neuromics. Cells were imaged in phase contrast mode with 40x objective.

Put our primary cells to the test!

MOR and NMDAR Interplay-Implications in Pain Control

2011-08-22T18:07:00.000-07:00

Our Opioid Receptor Antibodies continue to be referenced in publications by Pain Researchers. Many of these studies provide a greater understanding of how opioids alleviate pain and what modulates this ability.

For example, the capacity of opioids to alleviate inflammatory pain is negatively regulated by the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). This study drills down into the specifics of this regulation and references use of Neuromics' MOR1C Antibody: María Rodríguez-Muñoz, Pilar Sánchez-Blázquez, Ana Vicente-Sánchez, Esther Berrocoso and Javier Garzón. María Rodríguez-Muñoz, Pilar Sánchez-Blázquez, Ana Vicente-Sánchez, Esther Berrocoso and Javier Garzón. The Mu-Opioid Receptor and the NMDA Receptor Associate in PAG Neurons: Implications in Pain Control. Neuropsychopharmacology , (3 August 2011) | doi:10.1038/npp.2011.155.

Abstract: The capacity of opioids to alleviate inflammatory pain is negatively regulated by the glutamate-binding N-methyl-D-aspartate receptor (NMDAR). Increased activity of this receptor complicates the clinical use of opioids to treat persistent neuropathic pain. Immunohistochemical and ultrastructural studies have demonstrated the coexistence of both receptors within single neurons of the CNS, including those in the mesencephalic periaqueductal gray (PAG), a region that is implicated in the opioid control of nociception. We now report that mu-opioid receptors (MOR) and NMDAR NR1 subunits associate in the postsynaptic structures of PAG neurons. Morphine disrupts this complex by protein kinase-C (PKC)-mediated phosphorylation of the NR1 C1 segment and potentiates the NMDAR–CaMKII, pathway that is implicated in morphine tolerance. Inhibition of PKC, but not PKA or GRK2, restored the MOR–NR1 association and rescued the analgesic effect of morphine as well. The administration of N-methyl-D-aspartic acid separated the MOR–NR1 complex, increased MOR Ser phosphorylation, reduced the association of the MOR with G-proteins, and diminished the antinociceptive capacity of morphine. Inhibition of PKA, but not PKC, CaMKII, or GRK2, blocked these effects and preserved morphine antinociception. Thus, the opposing activities of the MOR and NMDAR in pain control affect their relation within neurons of structures such as the PAG. This finding could be exploited in developing bifunctional drugs that would act exclusively on those NMDARs associated with MORs.

I will continue to post these studies. They give hope for pain sufferers as many propose potential new druggable targets.

Plasma netrin-1 is a diagnostic biomarker of human cancers

2011-08-18T07:33:00.000-07:00

I am pleased to report broadening application for our Stem Cell Markers as a diagnostic for cancers. This publication references use of our Netrin-1 antibody.

Ganesan Ramesh, Arthur Berg, and Calpurnia Jayakumar. Plasma netrin-1 is a diagnostic biomarker of human cancers. Biomarkers. Author manuscript; available in PMC 2011 July 26. Published in final edited form as: Biomarkers. 2011 March; 16(2): 172–180. Published online 2011 February 8. doi: 10.3109/1354750X.2010.541564.
Objectives: To determine whether plasma netrin-1 can be used as a diagnostic biomarker of human cancer.

Materials and Methods: A total of 300 cancer plasma samples from breast, renal, prostate, liver, meningioma, pituitary adenoma, glioblastoma, lung, pancreatic and colon cancer patients were compared against 138 control plasma samples. Netrin-1 levels were quantified by ELISA and immunohistochemistry.

Results: Plasma netrin-1 levels were significantly increased in breast, renal, prostate, liver, meningioma, pituitary adenoma, and glioblastoma cancers as compared to control samples.

Discussion and Conclusion: Our results suggest that plasma netrin-1 can be used as a diagnostic biomarker for many human cancers.

Image: Immunohistochemical localization of netrin-1 in renal cell carcinoma (RCC) tissues. A. Secondary antibody control showing no staining. B. Normal adjacent tissues do not show any staining for netrin-1. C. Stage I RCC shows staining for netrin-1. D. Stage II RCC shows staining for netrin-1. E-F.

Netrin-1 Immunohistochemistry: Stage I–III renal cell carcinoma and normal tissue section (Tissue Array) was obtained from Biomax to immunolocalize netrin-1, as described previously (29). Briefly, tissue sections were dewaxed and rehydrated with graded ethanol (100%, 90%, 70% and 30%) and then washed with PBS. Antigen retrieval was carried out using citrate buffer and steamer. The tissue section was permeabilized with 0.2% Triton X-100 in PBS, and washed and blocked with PBS containing 5% donkey serum and 1% BSA. Primary antibodies included a chicken anti-netrin-1 polyclonal antibody (Neuromics cat # CH23002). Primary antibodies were detected using secondary antibodies conjugated with biotin, which was followed by incubation with streptavidin-horseradish peroxidase (Pierce). Slides were mounted in permount and photographed using an Olympus microscope attached to a CCD camera.
Potent diagnostic tools for cancers save lives. This is especially true of markers that can diagnose them in early stages. I will post these important studies as the cross my radar scope.

SOX2 and Initiation of Breast Tumors

2011-08-12T17:39:00.000-07:00

I consider it a feather in Neuromics' cap when our reagent(s) are referenced in a Nature Journal. More importantly it enables me to keep the pulse on novel and important discovery.

This pub references use of one of our SOX2 Antibody and comes from Dr. Angel García Martín and his Team at INBIOMED: O Leis, A Eguiara, E Lopez-Arribillaga, M J Alberdi, S Hernandez-Garcia, K Elorriaga, A Pandiella, R Rezola and A G Martin. Sox2 expression in breast tumours and activation in breast cancer stem cells. Oncogene , (8 August 2011) | doi:10.1038/onc.2011.338.

The important insight from this study is: "Over-expression of Sox2 increased mammosphere formation, effect dependent on continuous Sox2 expression; furthermore, Sox2 knockdown prevented mammosphere formation and delayed tumour formation in xenograft tumour initiation models. Induction of Sox2 expression was achieved through activation of the distal enhancer of Sox2 promoter upon sphere formation, the same element that controls Sox2 transcription in pluripotent stem cells. These findings suggest that reactivation of Sox2 represents an early step in breast tumour initiation, explaining tumour heterogeneity by placing the tumour-initiating event in any cell along the axis of mammary differentiation."

Could these findings ultimately lead to a better diagnostic for Breast Cancer? I'll keep you posted.

Lab Highlights: Breast cancer is the most frequent cancer in women making up to 20% of all tumours diagnosed in women, with 1 million new cases diagnosed every year worldwide (16,000 new cases only in Spain). Worlwide it causes over 350,000 deaths with an increasing tendency. Breast cancer stem cells show the phenotype CD44+/CD24low/-Lin- though only a fraction of this population has the capacity to initiate tumours. Therefore a complete and precise description of the breast cancer stem cells is lacking.

The focus of this laboratory is to identify, isolate and culture breast cancer stem cells from natural breast tumours and compare at the molecular level with normal mammary stem cells. This research involves the stablishment of both in vitro and in vivo functional assays and the molecular characterization (both genomic and proteomic) of breast cancer stem cells to define the mechanisms responsible for its transformed phenotype.

High Content and High Throughput Toxicity Screening

2011-08-07T08:06:00.000-07:00

Kits designed for Drug Discovery and Development

Our customers have been impressed with the capablities of our in vivo and in vitro apoptosis and toxicity kits. Here's a recent pub referencing use of one of our FLICA™ in vitro Caspase Kits: Giovanna Grandinetti, Nilesh P. Ingle, and Theresa M. Reineke. Interaction of Poly(ethylenimine)–DNA Polyplexes with Mitochondria: Implications for a Mechanism of Cytotoxicity. Mol. Pharmaceutics, Article ASAP Publication Date (Web): June 23, 2011 Copyright © 2011 American Chemical Society...inhibitor of caspases (FLICA) specific for caspase-9 (Neuromics, Inc)...
Images: Jurkat cells were treated with 1 µM staurosporine for 3 hours to induce caspase 9 activity (top), or were treated with a control (bottom). Both populations were incubated with ICT’s green FAM-LEHD-FMK FLICA™ caspase 9 reagent. DIC images were taken of both samples. Almost all cells in the induced sample (top) fluoresce green therefore they have activated caspase 9. None of the control cells (bottom) fluoresce green, therefore they do not have activated caspase 9 Courtesy of Dr. Brian Lee, ICT

Neuromics is pleased to announce the addition of HemoGenix® Predictive in vitro Toxicity and Apoptosis Kits:

Solutions for predictive in vitro toxicity and apoptosis are now important than ever. These kits enable you to do high throughput and high content screening of stem cells, progenitors and primary cells.
Kit options include:

•LumiSTEM™-96 iPS and LumiSTEM™-iPS HT Assays to Study Induced Pluripotent Stem Cells (iPS) and Toxicity to iPS Cells and Cells Derived from iPS Cells.

•LUMENESC™-Tox HT (LUMENESC™-96 Tox and LUMENESC™-384 HT). A Toxicity Screening and Testing Platform for Cells of the Mesenchymal Stem/Stromal Cell System.

•HALO®-Tox HT Predictive Hemotoxicity Platform using HALO®-96 Tox and HALO®-384 HT. A Highly Predictive, In Vitro Stem and Progenitor Cell Hemotoxicity Screening and Testing Platform for all Stages of Drug Development and Xenobiotic.

I will continue to post regarding progress.

TRPV1 and Diabetic Neuropathy

2011-07-30T08:49:00.000-07:00

Thermal hyperalgesia is a common sympton of Diabetic Periperal Neuropathy (DPN). It is one of most difficult types of pain to treat. The development of tolerance, inadequate relief and potential toxicity of classical antinociceptives warrant the investigation of the newer agents to relieve this pain.

The elevated expression of Transient receptor potential vanilloid 1 (TRPV1) suspected as a transmitter of this pain. Dr. Louis Premkumar and his team at SIU have recently published results that further demonstrate the role of TRPV1: Mahendra Bishnoi, Christine A Bosgraaf, Mruvil Abooj, Linlin Zhong, Louis S Premkumar. Streptozotocin-Induced Early Thermal Hyperalgesia is independent of Glycemic State of Rats: Role of Transient Receptor Potential Vanilloid 1(TRPV1) and inflammatory mediators. Molecular Pain 2011, 7:52 doi:10.1186/1744-8069-7-52. Published: 27 July 2011.

Figure 4. Altered TRPV1 staining in spinal cord dorsal horn of STZ-treated rats. A. Representative images of TRPV1 staining from a vehicle-treated, STZ-HG and STZNG rats. An enlarged segment has also been shown. B. Average gray values/10,000 μm² area of TRPV1 staining in dorsal horn was significantly increased (p<0.05) in both STZ-HG and STZ-NG rats as compared to vehicle-treated rats. Asterisk (*) represents p < 0.05. Scale bar is 200 μm and 50 μm for upper and lower panels, respectively.

Conclusions: From these results, it is concluded that TRPV1 is an integral component of initiating and maintaining inflammatory thermal hyperalgesia, which can be alleviated by intrathecal administration of RTX. Further, the results suggest that enhanced expression and inflammation-induced sensitization of TRPV1 at the spinal cord may play a role in central sensitization in STZ-induced neuropathy.

Therapies that downregulate or silence TRPV1 expression could be the key to better treatments for the Thermal Algesia cause by diabetes. I will keep you posted.

hNP1 Neural Progenitors Thawing+ Plating Protocol

2011-07-29T07:21:00.000-07:00

This is a great video that shows thawing + plating protocol for our eSC Derived Human NP1 Neural Progenitors.

Potent and Cost Effective Cell Based Assays

2011-07-27T09:13:00.000-07:00

I have had many conversations with basic and drug discovery researchers on improving cell based assays. Here's the wish list:

More potent cells/media
More accurate analytic tools-quatititative and reproducible results
Ability to use cells and tools in high throughput/high content screening.
Cost effectiveness

This wish list is front and center in determining the cells/media and related tools we add to Neuromics' offerings. We are pleased to announce the addition of our Hemogenix's Bioluminomics™ In-Vitro Cell Assays, MSCGro™ Mesenchymal Stem Cell Media and Umbilical Cord Blood derived hMesenchymal Stem Cells.

These provide quantitation, not subjectivity. It includes assay calibration and standardization. It means assay validation. It produces results you can trust and rely on. It means innovation and flexibility. It is advanced technology that is fast to learn, easy to use and above all, cost effective.

Assays options:

LumiSTEM™ for Primary Stem and Explanted Cells, Stem and Other Cell Lines-Selecting LumiSTEM™ Assays(pdf - 418Kb)
LUMENESC™ for Mesenchymal Stem/Stromal Cell System-Selecting LUMENESC™ Assays(pdf - 984Kb)
HALO® for for Human Lympho-Hematopoietic Stem and Progenitor Cell Research.-Selecting HALO™ Assays(pdf - 1.7M)
Biolumonics Basic Procedures(pdf - 323Kb)

Available Cells:

Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes
STEMEZ^TM Human Neural Progenitor Neuron Discovery Kits-Derived from H9 (WA09) ECSs-Consistent, Easy to Use & Cost Effective
Human Mesenchymal Stem Cells (hMSCs-hMSCs derived from pancreas and umbilical cord blood
Mammalian Cell Lines

Media:
STEMEZ(TM) hN2 Human Neurons Culture Media
MSCGro™ Mesenchymal Stem Cell Media
NbActiv4

I will continue to post customer input and related data on Neuromics' Cell Based Assay Tools.

Differential healing properties of human ACL and MCL Stem Cells

2011-07-23T14:39:00.000-07:00

Autologous Stem Cell therapies for human injury and disease are gaining momentum. Understanding the properties of Stem Cell Colonies that have potential for these therapies is key to optimizing treatments. This study provides knowledge on the properties and their impact on future therapies for anterior cruciate ligament (hACL) and medial collateral ligament (hMCL) of the knee joint.
Jianying Zhang, Tiffany Pan, Hee-Jeong Im, Freddie H Fu and James HC Wang. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity. BMC Medicine 2011, 9:68doi:10.1186/1741-7015-9-68.

Background: The (hACL) and medial collateral ligament (hMCL) of the knee joint are frequently injured, especially in athletic settings. It has been known that, while injuries to the MCL typically heal with conservative treatment, ACL injuries usually do not heal. As adult stem cells repair injured tissues through proliferation and differentiation, we hypothesized that the hACL and hMCL contain stem cells exhibiting unique properties that could be responsible for the differential healing capacity of the two ligaments.

Methods: To test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry.

Images:The expression of stem cell markers in hACL-SCs and hMCL-SCs. At passage 5, hACL-SCs had already become highly elongated in confluent culture, a typical fibroblast phenotype (A). In contrast, even at passage 13, confluent hMCL-SCs remained cobblestone-like (B). Moreover, hACL-SCs no longer expressed nucleostemin (C) or SSEA-4 (E) at passages > 5, whereas hMCL-SCs expressed both stem cell markers at passage 13 (D, F). Note, however, that hMCL-SCs at this high passage exhibited a lesser degree of nucleostemin expression compared to the cells at passage 1 (see Figure 3). The results shown here were obtained from a male donor of 27 years oldTo test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry.

Results: We found that both hACL stem cells (hACL-SCs) and hMCL stem cells (hMCL-SCs) formed colonies in culture and expressed stem cell markers nucleostemin and stage-specific embryonic antigen-4 (SSEA-4). Moreover, both hACL-SCs and hMCL-SCs expressed CD surface markers for mesenchymal stem cells, including CD44 and CD90, but not those markers for vascular cells, CD31, CD34, CD45, and CD146. However, hACL-SCs differed from hMCL-SCs in that the size and number of hACL-SC colonies in culture were much smaller and grew more slowly than hMCL-SC colonies. Moreover, fewer hACL-SCs in cell colonies expressed stem cell markers STRO-1 and octamer-binding transcription factor-4 (Oct-4) than hMCL-SCs. Finally, hACL-SCs had less multi-differentiation potential than hMCL-SCs, evidenced by differing extents of adipogenesis, chondrogenesis, and osteogenesis in the respective induction media.
Conclusions: This study shows for the first time that hACL-SCs are intrinsically different from hMCL-SCs. We suggest that the differences in their properties contribute to the known disparity in healing capabilities between the two ligaments.
I will be posting more on autologous stem cell therapies research.

Understanding Rett Syndrome Pathologies

2011-07-21T08:17:00.000-07:00

Dr Jeffrey Neul and his team at Baylor Medical College have been studying the root causes of pathologies associated with Rett Syndrome.

This disease is a neurodevelopmental disorder caused by mutations in methyl-CpG-binding protein 2 (MECP2), a transcriptional regulator. In addition to cognitive, communication, and motor problems, affected individuals have abnormalities in autonomic function and respiratory control. Sufferers often die young due to these abnormalities.

They found that MeCP2 is necessary within the brainstem and spinal cord for normal lifespan, normal control of heart rate, and respiratory response to hypoxia. Here's the exciting news: restoration of MeCP2 in a subset of the cells in this same region is sufficient to rescue abnormal heart rate and abnormal respiratory response to hypoxia. Furthermore, restoring MeCP2 function in neural centers critical for autonomic and respiratory function alleviates the lethality associated with loss of MeCP2: Christopher S. Ward, E. Melissa Arvidel, Teng-Wei Huang, Jong Yoo, Jeffrey L. Noebels, and Jeffrey L. Neul. MeCP2 Is Critical within HoxB1-Derived Tissues of Mice for Normal Lifespan. The Journal of Neuroscience, 13 July 2011, 31(28): 10359-10370; doi: 10.1523/JNEUROSCI.0057-11.2011

I will be keeping my finger of the pulse of Dr. Neul and team's research. It could be one of the keys that unlocks the door to creating theapies for Rhett Syndrome. This would be good news for sufferers and their loved ones. There is hope.

We would also like to thank the authors for referencing use of our goat polyclonal Islet-1 antibody.

Guinea Pig P2X3 Update-Good News

2011-07-06T19:07:00.000-07:00

I have had to say to many customers, "our guinea pig P2x3 is on backorder". The increasing number of pubs referencing this antibody only amped demand.

We tried and tried to re-make it. The result was none of the bleeds we tested had a signal strong enough to release the antibody. We had a customer suggest re-testing several of the more promising bleeds. Thank you! We have good news on results and we are offering for 50% off. This is to acknowledge the investment required for TSA and Guinea Pig Biotinylated Antibody.

Here're the recent pubs I referenced:

Gabriela Castañeda-Corral, Héctor I. Rocha-González, Beatriz Godínez-Chaparro, Juan Miguel Jiménez-Andrade and Vinicio Granados-Soto. Role of the spinal Na+/H+ exchanger in formalin-induced nociception. Neuroscience Letters. doi:10.1016/j.neulet.2011.06.048....SP (guinea pig; 1:500; Cat# GP14110; Neuromics), CGRP (goat, 1:500; Cat# Ab36001; Abcam) and P2X3 receptor (guinea pig: 1:10,000; Cat# GP10108; Neuromics)...
Anna M.W. Taylora and Alfredo Ribeiro-da-Silva. GDNF levels in the lower lip skin in a rat model of trigeminal neuropathic pain: Implications for nonpeptidergic fiber reinnervation and parasympathetic sprouting. PAIN Volume 152, Issue 7, July 2011, Pages 1502-1510. doi:10.1016/j.pain.2011.02.035.
...Sections were then incubated for 48h at 4°C with a guinea pig polyclonal anti-P2X3 (1:25,000; Neuromics, Edina, MN)...

Transfection/Infection of Primary Neurons

2011-06-20T07:04:00.000-07:00

Gene Expression Analysis of Neurons is an important tools in basic research and the study of neuropathologies. At the Neuromics' blog: "siRNA, DsiRNA and Plasmid Transfection Efficiency", I have posted many examples of successful tarnsfection of primary neurons and related cells using both our Transfection Kits/Reagents and others.

The other puzzle piece for these studies is having a fresh, pure and easy to use source of cells. Here, Neuromics has many options. These primary neurons and neural progenitors are widely referenced in key publications. Applications referenced include: transfection, pharmacology, electrophysiology, immunocytochemistry, and neuronal development studies.

This posting features infection of our e18 Primary Rat Combined Hippocampus, Cortex, and Ventricular Neurons using Nipah virus related components and HeV pseudotyped virions

Aparna Talekar, Antonello Pessi, and Matteo Porotto. Infection of primary neurons mediated by Nipah virus envelope proteins: Role of host target cells in antiviral action. J. Virol. doi:10.1128/JVI.00452-11.

Abstract: We have previously described heterotypic peptides from parainfluenza virus that potently inhibit Nipah virus in vitro, but are not efficacious in vivo. By contrast, our second-generation inhibitors, featuring a cholesterol moiety, are also efficacious in vivo. The difference between in vitro and in vivo results led us to investigate the basis for this discrepancy. Here we compare the activity of the compounds in standard laboratory cells and in cells relevant to the natural tropism of Nipah virus, i.e. primary neurons, and show that while our first generation inhibitors are poorly active in primary neurons, the cholesterol-conjugated compounds are highly potent. These results highlight the advantage of evaluating antiviral potency in cells relevant to natural host target tissue.

Customer Data: Transfection of functional HeV glycoproteins and infection with HeV pseudotyped virions.In order to establish the feasibility of carrying out the proposed experiments in primary neurons, we show (figure ) that our assays are amenable to use in primary neurons. In the experiment, Combined Hippocampus, Cortex, and Ventricular -E18 (Neuromics) were plated, and at 3 days were transfected with plasmids encoding HeV G/F as well as YFP. On the following day, these cells were infected with HeV or VSV pseudotyped viruses bearing RFP. In the figure, (A) the phase contrast photos show the differentiated neurons; (B) upon excitation for RFP, the red fluorescence indicates neurons infected by HeV pseudotyped virions; (C) upon excitation for YFP, and the green fluorescence shows the efficiency of transfection in neurons. This experiment indicates that the proposed experiments can be carried out in primary neurons, which are transfectable and infectable in our systems, and thus supports all the proposed aims. Data Courtesy of Dr. Matteo Porotto, Weill Cornell Medical College. Larger Image

We will continue to keep you updated.

Chronic Type II Diabetes Mellitus and Cardiovascular Disease

2011-06-17T17:45:00.000-07:00

Our Neuropeptide and Neuropeptide Receptors and Leptin and Leptin Receptor Antibdodies are frequently used to study pathologies and biology specific to Obesity and Diabetes.

Here's a new publication studying the relationship between diabetic neuropathy and altered neuropeptide Y and its receptor expression levels in myocardium and plasma.

Robina Matyal, Feroze Mahmood, Michael Robich, Hiliary Glazera, Kamal Khabbaza, Philip Hessa, Cesario Bianchia, Robert Hagberga, Shu-Xu Hua, and Frank W. Sellkea. Chronic type II diabetes mellitus leads to changes in neuropeptide Y receptor expression and distribution in human myocardial tissue. European Journal of Pharmacology. Volume 665, Issues 1-3, 31 August 2011, Pages 19-28.

Abstract: Neuropeptide Y is one of the most abundant neurotransmitters in the myocardium, and is known to influence cardiovascular remodeling. We hypothesized that diabetic neuropathy could possibly be associated with altered neuropeptide Y and its receptor expression levels in myocardium and plasma. Plasma neuropeptide Y levels in diabetic (n = 24, HgbA1c 7.9 ± 1.1%) and non-diabetic (n = 27, HgbA1c 5.8 ± 0.5%) patients undergoing cardiac surgery utilizing cardiopulmonary bypass were analyzed. Right atrial tissue of these patients was used to determine the expression of neuropeptide Y, the receptors 1–5, and leptin by immunoblotting, real-time PCR and immunofluorescence. Apoptosis signaling and endostatin and angiostatin were measured to determine the effects of leptin.

Plasma neuropeptide Y levels were significantly increased in patients with Type II diabetes mellitus as compared to non-diabetic patients (P = 0.026). Atrial tissue neuropeptide Y mRNA levels were lower in diabetic patients (P = 0.036). There was a significant up-regulation of myocardial Y2 and Y5 receptors (P = 0.009, P = 0.01 respectively) in the diabetic patients. Leptin, involved with apoptosis and angiogenesis, was down regulated in diabetic patients (P = 0.05). The levels of caspase-3, endostatin and angiostatin were significantly elevated in diabetic patients (P = 0.003, P = 0.008, P = 0.01 respectively). Y1 receptors were more likely to be localized within the nuclei of cardiomyocytes and vascular smooth muscle cells.

Neuropeptide expression is altered differentially in the serum and myocardium by diabetes. Altered regulation of this system in diabetics may be in part responsible for the decreased angiogenesis, increased apoptosis, and increased vascular smooth muscle proliferation leading to coronary artery disease and heart failure in this patient population.

SOX2 as a Marker for Melanoma

2011-06-15T18:18:00.000-07:00

I would like to post a new application for our SOX2 neural progenitor marker.

Alvaro C. Laga, Qian Zhan,Carsten Weishaupt, Jie Ma, Markus H. Frank, George F. Murphy. SOX2 and nestin expression in human melanoma: an immunohistochemical and experimental study. Experimental Dermatology. Volume 20, Issue 4, pages 339–345, April 2011. DOI: 10.1111/j.1600-0625.2011.01247.x.

SOX2 is an embryonic neural crest stem-cell transcription factor recently shown to be expressed in human melanoma and to correlate with experimental tumor growth. SOX2 binds to an enhancer region of the gene that encodes for nestin, also a neural progenitor cell biomarker. To define further the potential relationship between SOX2 and nestin, we examined co-expression patterns in 135 melanomas and 37 melanocytic nevi. Immunohistochemical staining in 27 melanoma tissue sections showed an association between SOX2 positivity, spindle cell shape and a peripheral nestin distribution pattern. In contrast, SOX2-negative cells were predominantly epithelioid, and exhibited a cytoplasmic pattern for nestin. In tissue microarrays, co-expression correlated with tumor progression, with only 11% of nevi co-expressing SOX2 and nestin in contrast to 65% of metastatic melanomas, and preliminarily, with clinical outcome. Human melanoma lines that differentially expressed constitutive SOX2 revealed a positive correlation between SOX2 and nestin expression. Experimental melanomas grown from these respective cell lines in murine subcutis and dermis of xenografted human skin maintained the association between SOX2-positivity, spindle cell shape, and peripheral nestin distribution. Moreover, the cytoplasmic pattern of nestin distribution was observed in xenografts generated from SOX2-knockdown A2058 melanoma cells, in contrast to the periperhal nestin pattern seen in tumors grown from A2058 control cells transfected with non-target shRNA. In aggregate, these data further support a biologically significant linkage between SOX2 and nestin expression in human melanoma.

Assembly and Maintenance of GABAergic Synapses

2011-06-12T07:51:00.000-07:00

Understanding the mechanisms underlying Axon Growth and Guidance is key to finding the root cause of neurological diseases and discovering potential therapies.

In this important study, researchers TrkB is required for the localization of an Ig superfamily cell adhesion molecule, Contactin-1, in Golgi and granule cells and the absence of Contactin-1 also results in deficits in inhibitory synaptic development. This demonstrates that TrkB controls the assembly and maintenance of GABAergic synapses and suggest that TrkB functions, in part, through promoting synaptic adhesion: TrkB (Tropomyosin-Related Kinase B) Controls the Assembly and Maintenance of GABAergic Synapses in the Cerebellar Cortex. The Journal of Neuroscience, February 23, 2011 • 31(8):2769 –2780 • 276

Contactin-1 IHC and WB

Images: Inactivation of TrkB kinase activity disrupts the localization of GABAergic synaptic proteins. A–I, Homozygous mice carrying TrkB F616A allele were treated with water or 1NMPP1 from P0 to P28 and analyzed at P28. The localization of GAD65 (green; B), GAD67 (green; E) and gephyrin (red; H ) in the IGL is reduced in TrkB F616A mice treated with 1NMPP1 compared with TrkB F616A mice treated with water (A, D, G). C, F, I, Quantification of the area ratio of GAD65:vGluT1 (C), GAD67:vGluT1 (F ) and gephyrin:vGluT1 expression (I) in control and 1NMPP1-treated mice. J–R, Homozygousmice carrying TrkB F616A allele were treated with water or 1NMPP1 from P30 to P50 and analyzed at P50. The localization of GAD65 (green; K ), GAD67 (green; N ) and gephyrin (red; Q) is reduced in TrkB F616A mice treated with 1NMPP1 compared with control (J, M, P). L, O, R, Quantification of the area ratio of GAD65:vGluT1 (L), GAD67:vGluT1 (O) and gephyrin:vGluT1 expression (R) in control and 1NMPP1-treated mice. Scale bar, 10 um.

LepRb-STAT3 Pathway and Obesity Research

2011-05-29T08:43:00.000-07:00

Neuromics' Hypothalumus Neurons, Leptin Antibodies and Recombinant Proteins are being increasingly used by researchers studying root causes of diabetes and obesity. I am pleased to update you on an important study from our friends at Shanghai Jiaotong University School of Medicine. This publication references use of our LepRb/OBRb Antibody.

Pei Wang, Feng-Jiao Yang, Hui Du, Yun-Feng Guan, Tian-Ying Xu, Xue-Wen Xu, Ding-Feng Su, and Chao-Yu Miao. Involvement of Leptin Receptor Long Isoform (LepRb)-STAT3 Signaling Pathway in Brain Fat Mass– and Obesity-Associated(FTO) Downregulation during Energy Restriction. © 2011 The Feinstein Institute for Medical Research, www.feinsteininstitute.org.Online address: http://www.molmed.org. doi: 10.2119/molmed.2010.00013.
Abstract: Obesity is an important risk factor for cardiovascular disease, diabetes and certain cancers. The fat mass– and obesity associated (FTO) gene is tightly associated with the pathophysiology of obesity, whereas the exact role of FTO remains poorly understood. Here, we investigated the alternations of FTO mRNA and protein expression in the peripheral metabolic tissues and the brain upon energy restriction (ER) and explored the involvement of the leptin signaling pathway in FTO regulation under ER status. ER decreased the FTO mRNA and protein expression in hypothalamus and brainstem but not in periphery. Using doubleimmunofluorescence staining, FTO was found to be colocalized with the leptin receptor long isoform (LepRb) in arcuate nucleus of hypothalamus and the nucleus of the solitary tract. In LepRb mutant db/db mice, the FTO downregulation in brain and body weight reduction induced by ER were completely abolished. The enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) induced by ER was also impaired in db/db mice. Moreover, leptin directly activated the STAT3 signaling pathway and downregulated FTO in in vitro arcuate nucleus of hypothalamus cultures and in vivo wild-type mice but not db/db mice. Thus, our results provide the first evidence that the LepRb-STAT3 signaling pathway is involved in the brain FTO downregulation during ER.

LepRB (CH14104) staining of rat brain sections

Images: Frozen brain sections were incubated with LepRb (clone number CH14014; chicken antirat) and FTO (rabbit antirat) antibodies and then incubated with Cy3-conjugated secondary antibody (goat antichicken, red) or FITC-conjugated secondary (goat antirabbit, red). Nuclei were stained by 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI). NTS, nucleus of the solitary tract.

I will continue to track these kind of studies closely. They build the foundation for potential Obesity resduction therapies. This would have a major impact on growing burden of world wide Health Costs.

bradykinin B2 or purinergic P2Y receptors and SNs

2011-05-27T07:03:00.000-07:00

Our Neurotransmission Research Antibodies are proving to be important tools in studying excitability and firing properties of sympathetic neurons (SNs).

In this study the authors probe the role of a particular nerve cell potassium current, called the M-current, in the control of neurotransmitter release, using the contraction rate of the co-cultured heart cells as a functional read-out of noradrenaline release. Using several drugs and receptor agonists, we manipulated the activity of M-current in the nerve cells, which were stimulated by nicotine, and monitored its effect on heart cell beating. We find that the M-type potassium current has a robust role in the control of noradrenaline release from the nerve cells, and in the response of the heart cells to increased beating frequency as a result:

Oleg Zaika, Jie Zhang, and Mark S. Shapiro. Functional role of M-type (KCNQ) K+ channels in adrenergic control of cardiomyocyte contraction rate by sympathetic neurons. J. Physiol., May 2011; 589: 2559 - 2568.
Abstract: M-type (KCNQ) K+ channels are known to regulate excitability and firing properties of sympathetic neurons (SNs), but their role in regulating neurotransmitter release is unclear, requiring further study. We sought to use a physiological preparation in which SNs innervate primary cardiomyocytes to evaluate the direct role of M-channels in the release of noradrenaline (NA) from SNs. Co-cultures of rat SNs and mouse cardiomyocytes were prepared, and the contraction rate (CR) of the cardiomyocyte syncytium monitored by video microscopy. We excited the SNs with nicotine, acting on nicotinic acetylcholine receptors, and monitored the increase in CR in the presence or absence of the specific M-channel opener retigabine, or agonists of bradykinin B2 or purinergic P2Y receptors on the SNs. The maximal adrenergic effect on the CR was determined by application of isoproterenol (isoprenaline). To isolate the actions of B2 or P2Y receptor stimulation to the neurons, we prepared cardiomyocytes from B2 receptor or P2Y2 receptor knock-out mice, respectively. We found that co-application of retigabine strongly decreased the nicotine-induced increase in CR. Conversely, co-application of bradykinin or the P2Y-receptor agonist UTP augmented the nicotine-induced increase in CR to about half of the level produced by isoproterenol. All effects on the CR were wholly blocked by propranolol. Our data support the role of M-type K+ channels in the control of NA release by SNs at functional adrenergic synapses on cardiomyocytes. We conclude that physiological receptor agonists control the heart rate via the regulation of M-current in SNs.

This research has implications for forwarding the understanding of heart pacing.

MCP-1 promotes lung inflammation

2011-05-17T05:38:00.000-07:00

Providing reaserchers Immune/Inflammatory Response Markers is a growing business for us. Publications referencing their use is a key validation that they can do the job.

Here's a recent pub that references use of our goat anti-mouse RAGE polyclonal antibody. More importantly, it studies the role of Monocyte chemoattractant protein 1 (MCP-1) in modulating inflammatory response in the lungs: Marieke A.D. van Zoelen, Marleen I. Verstege, Christian Draing, Regina de Beer, Cornelis van’t Veera, Sandrine Florquin, Paul Bresser, Jaring S. van der Zee, Anje A. te Velde, Sonja von Aulock and Tom van der Poll. Endogenous MCP-1 promotes lung inflammation induced by LPS and LTA. doi:10.1016/j.molimm.2011.04.001.

AgRP's Role in Energy Homeostasis

2011-05-08T09:38:00.000-07:00

This blog has featured various posting on the building blocks and involved in Energy Homeostasis. This is key to future therapies for Obesity and Diabetes.
Here researchers study: Yongheng Cao1, Masanori Nakata, Shiki Okamoto, Eisuke Takano, Toshihiko Yada, Yasuhiko Minokoshi, Yukio Hirata, Kazunori Nakajima, Kristy Iskandar, Yoshitake Hayashi, Wataru Ogawa, Gregory S. Barsh, Hiroshi Hosoda, Kenji Kangawa, Hiroshi Itoh, Tetsuo Noda, Masato Kasuga, Jun Nakae.PDK1-Foxo1 in Agouti-Related Peptide Neurons Regulates Energy Homeostasis by Modulating Food Intake and Energy Expenditure. PLoS ONE 6(4): e18324. doi:10.1371/journal.pone.0018324.

IHC Protocol: For immunofluorescence analyses, mice were transcardially perfused with saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline, pH 7.4 (PBS). The brains were dissected and immersed in 4% paraformaldehyde at 4°C overnight and then soaked in 30% sucrose overnight. Frozen, free-floating coronal sections (4 µm thick) were cut through the arcuate nucleus with a microtome (Leica Microsystems). The sections were washed extensively in PBS for 20 min to quench endogenous peroxidase activity. For double staining of PDK1 and AGRP, the sections were stained with a Renaissance Tyramide Signal Amplification kit (#NEL701, Perkin Elmer, Waltham, MA) according to the manufacturer's protocol. The primary antibodies were Ab-241 (Signalway Antibody, Pearland, TX) for PDK1 and GT15023 (Neuromics, Edina, MN) for AGRP; the secondary antibodies were Alexa FluorR 594 chicken anti-rabbit IgG and Alexa FluorR 488 donkey anti-goat IgG (Molecular Probes, Eugene, OR). For double staining of Foxo1 and AGRP, we used an anti-FOXO1A antibody (ab12161, abcamR, Cambridge, UK), respectively. For double staining of FLAG and AGRP, the sections were stained using a Renaissance Tyramide Signal Amplification kit according to the manufacturer's protocol. The primary antibodies were the OctA-Probe (D-8: sc-807, Santa Cruz Biotechnology, Inc, Santa Cruz, CA); the secondary antibodies were Alexa FluorR 594 chicken anti-rabbit IgG and Alexa FluorR 488 donkey anti-goat IgG (Invitrogen, Carlsbad, CA).

Images: Functional defects of AGRP neurons in AGRPPdk1−/− mice. (A) Numbers of AGRP positive cells in the arcuate nuclei of AGRPPdk1+/+ (gray bar) and AGRPPdk1−/− (blue bar) mice. AGRP cell counts indifferent regions of the arcuate nucleus showed different numbers of AGRP neurons in AGRPPdk1+/+ (n = 3) and AGRPPdk1−/− mice (n = 3). (B) Representative Immunofluorescence images of AGRP in the hypothalamic regions of AGRPPdk1+/+ (left panel) and AGRPPdk1−/− mice (right panel). Green, AGRP; blue, DAPI. Scale bars indicate 100 µm. (C) Expression in the fed-state of hypothalamic neuropeptide genes in control (gray bar) and AGRPPdk1−/−(blue bar) mice. Data were normalized to β-actin expression and represent the mean ± SEM of six mice per genotype.

doi:10.1371/journal.pone.0018324.g002

Key Findings:PDK1 and Foxo1 signaling pathways play important roles in the control of energy homeostasis through AGRP-independent mechanisms. data indicated that PDK1 was indispensable for the orexigenic activity of AGRP neurons. Hypothalamic AGRP neurons express AGRP, NPY, the neurotransmitter GABA, and potentially other undiscovered molecules. In AGRPPdk1−/− mice, the expression of Agrp and Npy tended to be lower than that observed in control mice although the difference was not significant. Interestingly, the Δ256Foxo1AGRPPdk1−/− mice exhibited significantly increased food intake compared to AGRPPdk1−/− mice in spite of significantly decreased expression of Agrp and Npy. Therefore, changes in the expression levels of Agrp and Npy may not explain the changes in food intake in AGRPPdk1−/− mice.

GFAP and Mouse Myenteric Plexus

2011-03-31T17:09:00.000-07:00

Our Neuronal-Glial Markers are important tools for our customers investigating expression in the CNS and PNS. I have posted images showing staining of mouse retinal astrocytes and in the ventral horn, funiculus of adult rat spinal cord and mouse medulloblastoma stem cells using our GFAP antibodies.

I wanted to share an excellent image generated by Dr. Kate Ellacott's lab at Vanderbilt University.

GFAP (Chicken-Cat#: CH22102) staining in of enteric glia in the myenteric plexus of the mouse gastrointestinal tract. Staining was performed in methanol/acetone fixed frozen sections using 1:1000 dilution of the antibody followed by 1:500 anti-chicken Alexa 594.

Related Reagents:

GFAP (Chicken-Cat#: CH23011)
GFAP (Mouse-Cat#; MO15052)
GFAP (Rabbit-Cat#:RA19063)
Neuronal-Glial Markers -Astrocytes, Glia, Microglia, Olidogodendrocytes, Progenitors and Schwann Cell.

STEMEZ hNP1 Neural Progenitors and Ion Channels

2011-03-23T18:25:00.000-07:00

In my conversation with neuro-drug discover researchers, I am frequently being asked about the potential of using our STEMEZ(TM) hNP1 Human Neural Progenitors Expansion Kits for studying ion channels. How effective are these cells as a source for studying neurodegenerative diseases and for drug screening assays? There is good news.

When differentiated, these neural progenitors express subunits of glutamatergic, GABAergic, nicotinic, purinergic and transient receptor potential receptors. In addition, sodium and calcium channel subunits were also expressed. Functionally, virtually all the NP cells exhibited delayed rectifier potassium channel currents and some differentiated cells exhibited tetrodotoxin sensitive, voltage-dependent sodium channel current under whole-cell voltage clamp and action potentials could be elicited by current injection under whole-cell current clamp. These results indicate that removing basic fibroblast growth factor from the neural progenitor cell cultures leads to a post-mitotic state, and also results in the capability to produce excitable cells that can generate action potentials. This is the first data demonstrating capabilitiesof these cells for ionotrophic receptor assays and ultimately for electrically active human neural cell assays for drug discovery.

Images: Glutamate receptor expression in hNP cells and differentiated hNP cells The expression of ionotropic glutamate receptors might also be an indicator of neuronal maturation. These receptors are composed of three distinct families: NMDA, kainate and AMPA receptors. The hNP cells and differentiated hNP cells cultured in the absence of bFGF for 2 weeks were analyzed for mRNA expression of subunits of each glutamate receptor subtype relative to hESCs. Significant increases (p<0.05) in Grin2b were seen in hNP cells (20 fold) and differentiated hNP cells (25 fold) relative to hESCs (Figure 3A). Additionally, Grin1 and Grin2d were significantly increased (p<0.05) only in differentiated hNP cells relative to hESCs, but not in undifferentiated hNP cells (Figure 3A). Of the kainate receptors, Grik4 and Grik5 were significantly (p<0.05) increased only in undifferentiated hNP cells relative to hESCs (Figure 3B); whereas, Grik2 was significantly (p<0.05) increased only in hNP cells where bFGF had been removed (Figure 3B). AMPA receptor subunits were also examined. Gria1 and Gria4 were up regulated in hNP cells relative to hESCs (Figure 3C). Two week differentiated hNP cells showed significant (p<0.05) up regulation of Gria2 and Gira4 relative to hESCs (Figure 3C). To determine if functional glutamate channels exist in differentiated hNP cells, calcium influx in response to AMPA, kainic acid or NMDA application was measured on hNP cells, 14 days after the removal of bFGF. Figure 3G indicates that NMDA could not depolarize differentiated or undifferentiated hNP cells enough to cause significant calcium influx above background. In contrast, AMPA and kainic acid can cause calcium influx which can be potentiated by AMPA receptor specific modulator, cyclothiazide (50 μM, Figure 3G).Calcium influx was detected in the presence of cyclothiazide in calcium activity as measured (Figure 3H).

Images: Sodium channel activity in differentiated hNP cells was measured using whole cell voltage clamp. 81 total hNP cells cultured in the absence of bFGF from 4 to 27 days were analyzed. Of these, 34 exhibited no fast inward currents in response to a step depolarization indicating the 348 absence of functional voltage gated sodium channels (Figure 4G). The remaining cells yielded between 0.04 - 1.5 nA of inward current in response to the step depolarization (Figures 4B and 4G). These currents inactivated rapidly in all cases (Figures 4B and 4C) and could be abolished with the addition of 1 μM TTX (n = 3 cells; Figure 4C). Voltage-dependent steady state inactivation (n = 11 cells; Figure 4D) and recovery from fast inactivation (n = 5 cells; Figure 4E) were also observed on several positive cells. A subset of these cells was subjected to current clamp and action potentials were elicited by current injection (n = 8 cells, Figure 4F). In support of this, increasing concentrations of a sodium channel activator veratridine in a FLIPR assay on differentiated hNP cells show an increasing calcium response (Figure 4H). This probably resulted from voltage-gated sodium channel depolarization of cells that subsequently allowed calcium influx through calcium channels. These data indicate that differentiation of hNP cells by removal of bFGF can lead to a neuronal cell that can generate action potentials and depolarize the cell. The 58% hit rate for voltage-gated sodium channel function (Figure 4G), does not reflect the true proportion of sodium channel positive cells in our differentiated hNP cells, but rather our ability to morphologically distinguish these cells from negative cells by eye. An example of the morphology of a sodium channel positive cell is shown in Figure 4A. The positive cells were phase bright with a few long processes.

pERK1/2, TRPV1 and Scalding Burn Pain

2011-03-15T14:06:00.000-07:00

I can only imaging the intense pain suffered by people with scalding pain injuries.

Understanding the bio-processes behind this form of nociceptive pain is a step towards finding better analgesics. In this study, the authors study several key markers for pain...phosphoERK1/2 and TRPV1 (surprisingly not a major piece of the this pain signaling process).

John P.M. Whitea, Chin Wing Koa, Antonio Rei Fidalgoa, Mario Cibellia, Cleoper C. Paulea, Peter J. Andersona, Celia Cruzb, Szabolcs Gombad, Klara Matesze, Gabor Veressd, Antonio Avelinob and Istvan Nagya. Severe burn injury induces a characteristic activation of extracellular signal-regulated kinase 1/2 in spinal dorsal horn neurons. European Journal of Pain.doi:10.1016/j.ejpain.2010.12.006...pERK1/2 (1:1000, Neuromics; RA15002)..
Abstract: We have studied scalding-type burn injury-induced activation of extracellular signal-regulated kinase 1/2 (ERK1/2) in the spinal dorsal horn, which is a recognised marker for spinal nociceptive processing. At 5 min after severe scalding injury to mouse hind-paw, a substantial number of phosphorylated ERK1/2 (pERK1/2) immunopositive neurons were found in the ipsilateral dorsal horn. At 1 h post-injury, the number of pERK1/2-labelled neurons remained substantially the same. However, at 3 h post-injury, a further increase in the number of labelled neurons was found on the ipsilateral side, while a remarkable increase in the number of labelled neurons on the contralateral side resulted in there being no significant difference between the extent of the labelling on both sides. By 6 h post-injury, the number of labelled neurons was reduced on both sides without there being significant difference between the two sides. A similar pattern of severe scalding injury-induced activation of ERK1/2 in spinal dorsal horn neurons over the same time-course was found in mice which lacked the transient receptor potential type 1 receptor (TRPV1) except that the extent to which ERK1/2 was activated in the ipsilateral dorsal horn at 5 min post-injury was significantly greater in wild-type animals when compared to TRPV1 null animals. This difference in activation of ERK1/2 in spinal dorsal horn neurons was abolished within 1 h after injury, demonstrating that TRPV1 is not essential for the maintenance of ongoing spinal nociceptive processing in inflammatory pain conditions in mouse resulting from at least certain types of severe burn injury.

TrkB and Assembly/Maintenance of GABAergic Neurons

2011-03-09T08:52:00.000-08:00

Publications that provide unique insight into neuro-and synptogenesis catch my attention.

This publication focuses on the mechanisms that direct inhibitory circuits. Specifically, the authors study assembly and maintenance of GABAergic inhibitory synapses between Golgi and granule cells in the mouse cerebellar cortex.

Albert I. Chen, Cindy N. Nguyen, David R. Copenhagen, Sylvia Badurek, Liliana Minichiello, Barbara Ranscht, and Louis F. Reichardt. TrkB (Tropomyosin-Related Kinase B) Controls the Assembly and Maintenance of GABAergic Synapses in the Cerebellar Cortex . The Journal of Neuroscience, 23 February 2011, 31(8): 2769-2780; doi: 10.1523/JNEUROSCI.4991-10.2011.

Our cell adhesion marker-Contactin-1 was referenced: Immunohistochemistry 1:1,000; western blot: 1:1,000

Conclusion Highlights: Kinase activity of TrkB is required not simply to initiate GABAergic synapse formation, but also to maintain these synapses in adulthood. We also show that the localization of Contactin-1 at the synaptic contacts between Golgi and granule cells requires TrkB suggesting that TrkB promotes synapse formation and maintenance, in part, by controlling the localization of cell adhesion molecules.

Related Reagents:

Contactin-1, FC Chimera Recombinant Protein
Contactin-4 Recombinant Protein
Caspr2 Antibody
Axon Growth and Guidance Research
Antibodies
Axonal Growth and Guidance Research
Recombinant Proteins
Oligodendrocyte or Oligodendroglial Markers
Primary Neurons and Astrocytes-Primary
human, rat and mouse neurons and astrocytes

Notch Signaling and Neurodegenerative Disc Disease

2011-02-21T12:33:00.000-08:00

Notch proteins play a role in stimulating growth of a variety of cells. This includes the proliferation of stem cells.

In this study the investigators link hypoxia to notch signaling and if notch plays a role in intervertebral disc cell proliferation.

This publications also references use of our Notch3 antibody.

Akihiko Hiyama, Renata Skubutyte, Dessislava Markova, D. Greg Anderson, Sanjay Yadla, Daisuke Sakai, Joji Mochida, Todd J. Albert, Irving M. Shapiro, Makarand V. Risbud. Hypoxia activates notch signaling pathway in cells of the intervertebral disc: Implications in degenerative disc disease. DOI: 10.1002/art.30246. Copyright © 2011 by the American College of Rheumatology.
Results: Nucleus pulposus (NP) and annulus fibrosus (AF) cells expressed components of the notch signaling pathway. Notch2 expression was higher than the other notch receptors in both AF and NP. In both tissues, hypoxia increased notch1 and notch4 mRNA expression. In the AF, expression of the notch ligand, jagged1 was induced by hypoxia; in both the tissues, jagged2 expression was hypoxia-sensitive. To investigate if hypoxia promoted notch signaling, we measured the activity of twonotch responsive luciferase reporters, 12xCSL and CBF-1. L685458, a notch signaling inhibitor blocked hypoxic induction of both the reporter activities. We then investigated regulation of expression of the notch target gene Hes1. Hes1 expression was induced in hypoxia while co-expression of notch-ICD increased Hes1 promoter activity. Moreover, inhibition of notch signaling blocked disc cell proliferation. Finally, analysis of human discal tissues showed that there was increased expression of notch signaling proteins in the degenerate state.

Conclusion: In disc cells, hypoxia promotes expression of notch signaling proteins. Notch signaling is important to maintain proliferation of disc cells and hence offers a therapeutic target to restore cell number during degenerative disc disease.

Hope for sufferers of degenerative disease disc disease.

H9-WA09 Derived Human Neural Progenitors and Neurons Pricing

2011-02-12T07:56:00.000-08:00

Neuromics offers stable, potent and well characterized STEMEZ (TM) Human Neural Progenitor & Neuron Discovery Kits. These are derived from NIH registered human ES cell line H9 (WA09).

Details on the capabilities of these cells are detailed in a variety of publications. They work! As a result, I am trying to make it easier to justify using them when and where they are needed. In addition to having frequent updates on new references, methods and data, I want to offer the best pricing possible.

In researching pricing of cells derived from the same parental line and having similar characteristics, I noticed prices ranging from 995 to 2800 USD. Our pricing starts at 695 USD and we offer deeper discounting if required. I do not want price to be a barrier. Here's some sample data on these cells.

Images: Neural phenotypes derived from hN2 cell lines. (A) Phase contrast image of differentiated culture. (B) Network including post-mitotic motoneurons (HB9). (C) Cholinergic neuron. (D) Tuj-1 positive cells that are DAT-positive (dopamine transporter; closed arrow) and DAT-negative (open arrow). (E) Gabaergic neurons, inset illustrates GABA in axon, but not the dendrites (arrow).

G-protein Coupled Receptor Expression Patterns Are Altered as Human Embryonic Stem Details-From Poster Presented at Neuroscience 2008 by Dr. Steve Stice et al.
hN2 Cells-Electro Phys Data.

More on DOR

2011-02-07T14:09:00.000-08:00

I am always delighted when we recieve positive feedback on one of our new reagents. It is even better when a researcher graciously shares images. I would like to thank Dr. Prof. Dr. Rapheal Sell of Freie Universität Berlin for sharing these excellent Immunofluorescence images:

Images: IF detection of delta-opioid receptors in tissue engineered human oral mucosa models using Delta Opioid Receptor 361-372. The tissues were frozen at -80°C, cut into 8 µm slices and fixed with formaldehyde solution. Blocking was performed with goat serum (dilutet 1:20 in PBS) for 30 minutes at RT. Primary antibody dilutions in PBS/BSA/Tween-20 were 1:500, 1:1000 and 1:1500, slides were incubated overnight at 4°C. Secondary (fluorescent) antibody was diluted 1:400, slides were incubated for 30 minutes at room temperature in the dark. Sections were mounted with antifading mounting medium DAPI (blue) and evaluated by fluorescent microscopy

Related Reagents:

Delta Opioid Receptor 3-17

Delta Opioid Receptor 358-372

All Opioid Receptor Antibodies

Neurotransmission Research Antibodies-GPCRs, Ligand Gated Ion Channels, Biogenic Amines and more

Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes

PINK1 and Alzheimer's and Multiple Sclerosis

2011-01-26T17:19:00.000-08:00

I would like to highlight a recent publication referencing use of our PTEN-induced kinase/PINK1 Antibody.

Jack Van Horssen et al. investigated PINK1 expression in well-characterized brain samples derived from MS and AD individuals using immunohistochemistry.

Micha M.M. Wilhelmus, Susanne M.A. van der Pol, Quentin Jansen, Maarten E. Witte, Paul van der Valk, Annemieke J.M. Rozemuller, Benjamin Drukarch, Helga E. de Vries and Jack Van Horssen. Association of Parkinson disease-related protein PINK1 with Alzheimer disease and multiple sclerosis brain lesions. Free Radical Biology and Medicine. Volume 50, Issue 3, 1 February 2011, Pages 469-476.

Abstract: Mitochondrial dysfunction and oxidative stress are hallmarks of various neurological disorders, including multiple sclerosis (MS), Alzheimer disease (AD), and Parkinson disease (PD). Mutations in PINK1, a mitochondrial kinase, have been linked to the occurrence of early onset parkinsonism. Currently, various studies support the notion of a neuroprotective role for PINK1, as it protects cells from stress-mediated mitochondrial dysfunction, oxidative stress, and apoptosis. Because information about the distribution pattern of PINK1 in neurological diseases other than PD is scarce, we here investigated PINK1 expression in well-characterized brain samples derived from MS and AD individuals using immunohistochemistry. In control gray matter PINK1 immunoreactivity was observed in neurons, particularly neurons in layers IV–VI. Astrocytes were the most prominent cell type decorated by anti-PINK1 antibody in the white matter. In addition, PINK1 staining was observed in the cerebrovasculature. In AD, PINK1 was found to colocalize with classic senile plaques and vascular amyloid depositions, as well as reactive astrocytes associated with the characteristic AD lesions. Interestingly, PINK1 was absent from neurofibrillary tangles. In active demyelinating MS lesions we observed a marked astrocytic PINK1 immunostaining, whereas astrocytes in chronic lesions were weakly stained. Taken together, we observed PINK1 immunostaining in both AD and MS lesions, predominantly in reactive astrocytes associated with these lesions, suggesting that the increase in astrocytic PINK1 protein might be an intrinsic protective mechanism to limit cellular injury.

Immunohistochemistry

Immunohistochemistry was used to detect PINK1 immunostaining in temporal neocortex and white matter in AD, MS, and control subjects. Cryosections (5 μm) were air-dried and fixed in acetone for 10 min. Next, sections were incubated with an affinity-purified rabbit anti-PINK1 antibody (1:100; Neuromics, Edina, MN, USA) for 60 min at room temperature. Then, the slides were incubated with EnVision kit horseradish peroxidase-labeled anti-mouse/rabbit (DAKO, Glostrup, Denmark) for 30 min at room temperature and finally diaminobenzidine tetrachloride. Between incubation steps, sections were thoroughly washed with phosphate-buffered saline (PBS). After a short rinse in tap water sections were incubated with hematoxylin for 1 min and extensively washed with tap water for 10 min. Finally, sections were dehydrated with ethanol followed by xylol and mounted with Entellan (Merck, Darmstadt, Germany). All antibodies were diluted in PBS containing 0.1% bovine serum albumin (Boehringer–Mannheim, Germany), which also served as a negative control. Negative controls were essentially blank.

Image: In active lesions PINK1 immunostaining was intense in reactive astrocytes (arrows). Double labeling of PINK1 (green) with the astrocytic marker GFAP (red) demonstrated PINK1 expression in astrocytes (inset).

Related Reagents:

Neurodegenerative Disease Research
Antibodies

Neurodegenerative Disease Research Proteins

Neurotransmission
-Neurotransmission Research Antibody Categories

Neurotrophins and Growth Factor Antibodies

Neuron-Glial Expressed-Includes
Neurotrophin Proteins

Primary Neurons and Astrocytes
-Primary human, rat and mouse neurons and astrocytes by Category

Upregulations of P2X3 and ASIC3 involve in hyperalgesia

2011-01-15T19:57:00.000-08:00

It gets my attention when several of our ion channel markers are referenced in the title of a publication.

Kiyomi Hori, Noriyuki Ozaki, Shigeyuki Suzuki, Yasuo Sugiura. Upregulations of P2X3 and ASIC3 involve in hyperalgesia induced by cisplatin administration in rats. PAIN 149 (2010) 393–405

Findings: "We explored the role of ion channels expressed in DRG neurons in the painful neuropathy associated with cisplatin administration. Upregulations of TRPV2, P2X3 and ASIC3 may play important roles in the mechanical hyperalgesia induced by cisplatin. In addition to cutaneous hyperalgesia, cisplatin treatment might also induce muscle hyperalgesia associated with upregulations of P2X3 and
ASIC3. Interfering with these channels may prove to be a promising therapeutic target for treating painful symptoms of cisplatin neuropathy, and may further be able to ensure the continuation of anticancer therapy."

Images: ASIC3 (Dilution 1:10) amd P2X3 (Dilution 1:500) satining of rat Dorsal Root Ganglia (DRGs) of cisplatin-treated animals. After dilution in 0.1 M phosphate-buffered saline (PBS) containing 1.5% normal goat serum and 0.3% Triton X-100 (Sigma), DRG sections were incubated with either guinea pig polyclonal antiserum against synthetic rat ASIC3 and rabbit polyclonal antiserum against synthetic rat P2X3 (1:500; Neuromics). The sections for ASIC3 were reacted with reagents for 2 days at room temperature and others at 4^OC. After being rinsed with 0.1 M PBS, the sections were reacted in PBS with fluorescein-isothiocyanate (FITC)-conjugated goat anti-guinea pig or - rabbit IgG antibody (Vector Laboratories, Burlingame, CA, USA) at a concentration of 1:100. After being rinsed with 0.1 M PBS, the sections were cover-slipped in mounting medium (Immunon, Pittsburgh, PA, USA) and examined under a fluorescence microscope equipped with a digital camera

SOX 17 Antibody that Rocks

2011-01-11T05:24:00.000-08:00

Our SOX Antibodies are proving to be excellent Stem Cell Markers. I would like to highlight a pub that references use of our Sox17 and a variety of other markers:

Pere Santamaria, Ignacio Rodríguez-Pizá, Xavier Clemente-Casares, Jun Yamanouchi, Lola Mulero-Perez, Trond Aasen, Angel Raya1 and Juan Carlos Izpisúa Belmonte. Turning Human Epidermis Into Pancreatic Endoderm. The Review of DIABETIC STUDIES 159 Vol. 7-No. 2-2010. DOI 10.1900/RDS.2010.7.158.

This pub references use of a variety of markers for showing differentiation of Human embryonic stem (hES)and induced pluripotent stem (iPS)into into pancreatic endoderm structures.

Real-time PCR and flow cytometry. A: Changes in the levels of different mRNAs in undifferentiated, and progressively differentiated, hES and iPS cells. A series of plates were cultured, as described in the materials and methods section. These were used for RNA extraction at the end of each differentiation stage, immediately prior to change in culture conditions (days 1, 3, 7, 10, and 13). B: Flow cytometric analysis of Sox17 and FoxA2 expression on cells harvested on day 3.

Great Apoptosis Video

2011-01-08T06:31:00.000-08:00

Our Primary Hippocampal, Cortical and Ventricular Neurons in Action

2010-12-21T09:52:00.000-08:00

I would like to thank Dr. Lidia Gardner of University of Tennessee HSC for providing excellent images of cultures using our Combined Hippocampal, Cortical, and Ventricular Neurons.

View Larger Image

Here's her feedback: "I got 10 million cells total after extraction from the tissue. At Day 4 they all developed long axons. Thank you so much for the replacement."
Related Reagents:

STEMEZ(TM) hN2 Human Neurons Discovery Kit
E18 and E20 Rat Primary Neuronal Tissue -NEURON CULTURES
E18 Rat Primary Neuronal Tissue - ASTROCYTE CULTURES
E18 Mouse Neuronal Tissue -NEURON CULTURES
E18 Mouse Neuronal Tissue -ASTROCYTE CULTURES
Frozen Primary Rat Cortical Neurons
Human Brain Tissue (Blocks, Lysates and Slides)-New
Growth Media, Poly-D-Lysine Coverslips and Fluoro-Tracer
FluoGreen Tracer
Neuronal-Glial Markers-Astrocytes, Glia, Microglia, Olidogodendrocytes, Progenitors and Schwann Cell Markers
Neuron-Glial Expressed Proteins-Includes Neurotrophin Proteins

Markers for Medial Superior Olivary Neurons

2010-12-16T04:12:00.000-08:00

This is an excellent reference for researchers looking for immunohistochemistry images of slice preparations of the Neurons in the medial superior olive (MSO). It also references use of our widely used and frequently published MAP2 (Microtubule associated protein 2).

Kiri Couchman, Benedikt Grothe and Felix Felmy. Medial Superior Olivary Neurons Receive Surprisingly Few Excitatory and Inhibitory Inputs with Balanced Strength and Short-Term Dynamics. The Journal of Neuroscience, December 15, 2010, 30(50):17111-17121; doi:10.1523/JNEUROSCI.1760-10.2010.

Summary: Neurons in the medial superior olive (MSO) process microsecond interaural time differences, the major cue for localizing low-frequency sounds, by comparing the relative arrival time of binaural, glutamatergic excitatory inputs. This coincidence detection mechanism is additionally shaped by highly specialized glycinergic inhibition. Traditionally, it is assumed that the binaural inputs are conveyed by many independent fibers, but such an anatomical arrangement may decrease temporal precision. Short-term depression on the other hand might enhance temporal fidelity during ongoing activity. For the first time we show that binaural coincidence detection in MSO neurons may require surprisingly few but strong inputs, challenging long-held assumptions about mammalian coincidence detection. This study exclusively uses adult gerbils for in vitro electrophysiology, single-cell electroporation and immunohistochemistry to characterize the size and short-term plasticity of inputs to the MSO. We find that the excitatory and inhibitory inputs to the MSO are well balanced both in strength and short-term dynamics, redefining this fastest of all mammalian coincidence detector circuits.

Related Reagents:
Neuronal-Glial Markers-Astrocytes, Glia,
Microglia, Olidogodendrocytes, Progenitors and Schwann Cell Markers
Stem Cell Research Antibodies
Stem Cell Research Reagents
Primary Neurons and Astrocytes-Primary

human, rat and mouse neurons and astrocytes.

Fragile-X, Astrocytes and BMC Image of the Month

2010-11-27T04:43:00.000-08:00

Dr. Laurie Doering and his team at McMaster University are discovering root causes of Fragile X Syndrome. A disease manifested by cognitive impairment, attention deficit and autistic behaviours.

I wanted to share highlights and links to a recent publication as it contains interesting conclusions and some of the best multiple label staining of combined embryonic rat and mouse neurons-astrocytes cultures I have seen. No wonder that this is a highly accessed Biomed Central Article and includes the image of the month. The featured image references use of our MAP-2 antibody.

Shelley Jacobs , Meera Nathwani and Laurie C Doering. Fragile X astrocytes induce developmental delays in dendrite maturation and synaptic protein expression. BMC Neuroscience 2010, 11:132doi:10.1186/1471-2202-11-132.

Conclusions: These experiments are the first to establish a role for astrocytes in the delayed growth characteristics and abnormal morphological features in dendrites and synapses that characterize the Fragile X syndrome.

Image: Co-culture of embryonic mouse hippocampal neurons and astrocytes. Primary embryonic hippocampal neurons at 7 days in vitro, were stained with Microtubule Associated Protein-2 (MAP, green) to enable the visualization of the dendritic arbors. These neurons were cultured on top of a monolayer of primary cortical astrocytes, stained with an antibody directed against Glial Fibrillary Acidic Protein (GFAP, red). The cell nuclei were visualized by staining with 4',6-diamidino-2-phenylindole (DAPI, blue).

Related Links:
Neuronal-Glial Markers-Astrocytes, Glia, Microglia, Olidogodendrocytes, Progenitors and Schwann Cell Markers
Neurofilament or NF Antibodies
Stem Cell Research Antibodies
Stem Cell Research Reagents

Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes.

Cancer-Induced Bone Pain

2010-11-18T05:59:00.000-08:00

Bone crushing pain. This describes pain of the highest order. Our friend, Dr. Joseph Ghilardi, VAMC-Mpls. and his colleague, Dr. Patrick Manthy are finding the root causes of the intense and growing pain suffered by Cancer Victims. Here are highlights of a recent study:

Pain frequently accompanies cancer. What remains unclear is why this pain frequently becomes more severe and difficult to control with disease progression. Here we test the hypothesis that with disease progression, sensory nerve fibers that innervate the tumor-bearing tissue undergo a pathological sprouting and reorganization, which in other nonmalignant pathologies has been shown to generate and maintain chronic pain. Injection of canine prostate cancer cells into mouse bone induces a remarkable sprouting of calcitonin gene-related peptide (CGRP+) and neurofilament 200 kDa (NF200+) sensory nerve fibers. Nearly all sensory nerve fibers that undergo sprouting also coexpress tropomyosin receptor kinase A (TrkA+). This ectopic sprouting occurs in sensory nerve fibers that are in close proximity to colonies of prostate cancer cells, tumor-associated stromal cells and newly formed woven bone, which together form sclerotic lesions that closely mirror the osteoblastic bone lesions induced by metastatic prostate tumors in humans. Preventive treatment with an antibody that sequesters nerve growth factor (NGF), administered when the pain and bone remodeling were first observed, blocks this ectopic sprouting and attenuates cancer pain. Interestingly, reverse transcription PCR analysis indicated that the prostate cancer cells themselves do not express detectable levels of mRNA coding for NGF. This suggests that the tumor-associated stromal cells express and release NGF, which drives the pathological reorganization of nearby TrkA+ sensory nerve fibers. Therapies that prevent this reorganization of sensory nerve fibers may provide insight into the evolving mechanisms that drive cancer pain and lead to more effective control of this chronic pain state.

Image: Image:Shows rat mixed neuron/glial cultures stained with mouse monoclonal antibody to neurofilament subunit NF-L clone 7D1 (green) and chicken antibody to neurofilament NF-H. This antibody binds primarily to the phosphorylated axonal forms of NF-H, in contrast to the NF-L antibody which stains both axonal and dendritic/perikaryal neurofilaments. The NF-L antibody therefore reveals a prominent cell body in green, while the surrounding axonal profiles are orange, since the are bound by both NF-L and the chicken NF-H antibody. Blue is a DNA stain. Protocol on data sheet.

Juan M. Jimenez-Andrade, Aaron P. Bloom, James I. Stake, William G. Mantyh, Reid N. Taylor, Katie T. Freeman, Joseph R. Ghilardi, Michael A. Kuskowski, and Patrick W. Mantyh Pathological Sprouting of Adult Nociceptors in Chronic Prostate Cancer-Induced Bone Pain. J. Neurosci., Nov 2010; 30: 14649 - 14656 ; doi:10.1523/JNEUROSCI.3300-10.2010
Here're several other pubs referencing use of our antibodies in studying bone cancer pain:

Kyle G. Halvorson, BA, Molly A. Sevcik, BA, Joseph R. Ghilardi, BS, BA, Lucy J. Sullivan, BA, Nathan J. Koewler, BS, Frieder Bauss, PhD, and Patrick W. Mantyh, PhD. Intravenous Ibandronate Rapidly Reduces Pain, Neurochemical Indices of Central Sensitization, Tumor Burden, and Skeletal Destruction in a Mouse Model of Bone Cancer. Published online 2008 April 14. doi: 10.1016/j.jpainsymman.2007.10.005
...pro-dynorphin (DYN, polyclonal guinea pig anti-rat, 1:1,000; Neuromics, Minneapolis, MN)...

Timothy K. Y. Kaan, Ping K. Yip, Sital Patel, Meirion Davies, Fabien Marchand, Debra A. Cockayne, Philip A. Nunn, Anthony H. Dickenson, Anthony P. D. W. Ford, Yu Zhong, Marzia Malcangio, and Stephen B. McMahon Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats. Brain, September 2010; 133: 2549 - 2564.
......Slides were then incubated with rabbit anti-P2X3 (1:2000, Neuromics) and sheep anti-calcitonin gene-related peptide (1:1000, Biomol...anti-beta-III-tubulin (1:4000, Promega) and guinea pig anti-P2X3 (1:100, Neuromics). The next day, after three washes with phosphate-buffered......

I will keep you posted on this important topic.

Our TRPV1 Antibodies Rock

2010-11-10T06:10:00.000-08:00

We now have 40 publications referencing use of our TRP Antibodies in multiple applications. Here are the October-November 2010 publications referencing use of these antibodies:

VR1 N-Terminus (TRPV1)-Rabbit

T. Wu, L. Song, X. Shi, Z. Jiang, J. Santos-Sacchi and A.L. Nuttal. Effect of capsaicin on potassium conductance and electromotility of guinea pig outer hair cell. doi:10.1016/j.heares.2010.10.010
...anti-TRPV1 (rabbit polyclonal, RA10110, Neuromics, Edina, MN, USA) diluted to 1:500 with 1% BSA-PBS...antibody (TRPV-1) and its blocking peptide (104 M) (Neuromics, Edina, MN, USA)...

(TRPV1) - mouse specific
Julie A. Christianson, Klaus Bielefeldt, Sacha A. Malin and Brian M. Davis. Neonatal colon insult alters growth factor expression and TRPA1 responses in adult mice. Pain Volume 151, Issue 2, November 2010, Pages 540-549.
...primary antiserum to TRPV1 (1:4000; Neuromics, Minneapolis, MN; cat# RA14113)...

VR1 C-terminus (TRPV1)-Guinea Pig
N. Schuelert, C. Zhang, A.J. Mogg, L.M. Broad, D.L. Hepburn, E.S. Nisenbaum, M.P. Johnson and J.J. McDougal. Paradoxical effects of the cannabinoid CB2 receptor agonist GW405833 on rat osteoarthritic knee joint pain. Osteoarthritis and Cartilage Volume 18, Issue 11, November 2010, Pages 1536-1543.
...primary antiserum to TRPV1 (1:4000; Neuromics, Minneapolis, MN; cat# RA14113)...

Mariusz Mucha, Lezanne Ooi, John E. Linley, Pawel Mordaka, Carine Dalle, Brian Robertson, Nikita Gamper, and Ian C. Wood Transcriptional Control of KCNQ Channel Genes and the Regulation of Neuronal Excitability.
J. Neurosci., Oct 2010; 30: 13235 - 13245 ; doi:10.1523/JNEUROSCI.1981-10.2010
...1:1000 guinea pig anti-TRPV1 (Neuromics)...

Related Reagents:
All TRP Antibodies
Pain and Inflammation Research Antibodies
Neurotransmission -Neurotransmission Research Antibody Categories

Vitamin A Deficiency and Hirschsprung Disease

2010-10-28T16:02:00.000-07:00

Dr. Robert Heuckeroth and his team and Washington University recently published more results on the link between maternal Vitamin A Deficiency and Hirschsprung Disease. It underscores the importance of maternal vitamin A nutrition for preventing the diease penetrance and expressitivity:
Ming Fu, Yoshiharu Sato, Ariel Lyons-Warren, Bin Zhang, Maureen A. Kane, Joseph L. Napoli and Robert O. Heuckeroth. Vitamin A facilitates enteric nervous system precursor migration by reducing Pten accumulation. Development 137, 631-640 (2010) doi:10.1242/dev.040550.
SUMMARY

Hirschsprung disease is a serious disorder of enteric nervous system (ENS) development caused by the failure of ENS precursor migration into the distal bowel. We now demonstrate that retinoic acid (RA) is crucial for GDNF-induced ENS precursor migration, cell polarization and lamellipodia formation, and that vitamin A depletion causes distal bowel aganglionosis in serum retinolbinding-protein-deficient (Rbp4–/–) mice. Ret heterozygosity increases the incidence and severity of distal bowel aganglionosis induced by vitamin A deficiency in Rbp4–/– animals. Furthermore, RA reduces phosphatase and tensin homolog (Pten) accumulation in migrating cells, whereas Pten overexpression slows ENS precursor migration. Collectively, these data support the hypothesis that vitamin A deficiency is a non-genetic risk factor that increases Hirschsprung disease penetrance and expressivity, suggesting that some cases of Hirschsprung disease might be preventable by optimizing maternal nutrition.

By the way, the lab has been an ongoing consumer of our Ret Antibody and referenced use of this antibody in the publication.

Image: E12.5 mouse mid-gut slices were cultured to allow crest-derived cells to migrate onto the dish in response to GDNF. Cultures were maintained for 16 hours without added retinoic acid.

Related Reagents:

Ret (C-Terminus Fused)
Ret-Fluorescein Labeled
Ret-Allophycocyanin Labeled
Ret-Phycoerythrin Labeled
Neurotrophins and Growth Factor Antibodies
Neurotrophins-Neuron/Glial Marker
Recombinant Proteins
Neuron/Glial Markers
Stem Cell Research Reagents

Stems Cells and SCI

2010-10-21T07:01:00.000-07:00

This is fascinating and encouraging to sufferers of Spinal Cord Injury:

ScienceDaily (Oct. 9, 2010) — Researchers at Karolinska Institutet have shown how stem cells, together with other cells, repair damaged tissue in the mouse spinal cord. The results are of potential significance to the development of therapies for spinal cord injury: http://www.sciencedaily.com/releases/2010/10/101008082736.htm#

Enhancing Neurite Outgrowth

2010-10-09T17:10:00.000-07:00

We are pleased to announce the addition of new proteins to our Axon Growth and Guidance category. These are designed to enhance neurite out growth.

Name	Catalog #	Type	Species	Size	Price
NGF-b, NSO Derived	PR15083	Protein	H; M; R	100 ug	$285
NGF-b, NSO Derived, CF	PR15084CF	Protein	H; M; R	100 ug	$285
S100A13, CF	PR15085CF-50	Protein	H; R	50 ug	$315
Slit1	PR15075-50	Protein	Ch; H	50 ug	$315
Slit2, CHO	PR15085-50	Protein	Ch; H	50 ug	$315

Image: Cultured chick dorsal root ganglion neurons were grown in the presence of recombinant human NGF-b with (A) or without (B) recombinant mouse Slit2. The presence of the Slit2 protein signifi cantly enhanced neurite outgrowth.

Glutaredoxin 2 prevents aggregation of mutant SOD1

2010-09-22T14:22:00.000-07:00

Our PTEN-induced kinase, PINK1 or PARK6 Antibody is an excellent marker for Amyotrophic Lateral Sclerosis (ALS) and Parkinson's Disease (PD) researchers.

Here's new publication referencing use of this antibody: Alberto Ferri, Paolo Fiorenzo, Monica Nencini, Mauro Cozzolino, Maria Grazia Pesaresi, Cristiana Valle, Sara Sepe, Sandra Moreno, and Maria Teresa Carrì. Glutaredoxin 2 prevents aggregation of mutant SOD1 in mitochondria and abolishes its toxicity.
Hum. Mol. Genet., first published on Sep 20, 2010 as doi: doi:10.1093/hmg/ddq383

Abstract:
Vulnerability of motoneurons in amyotrophic lateral sclerosis (ALS) arises from a combination of several mechanisms, including protein misfolding and aggregation, mitochondrial dysfunction and oxidative damage. Protein aggregates are found in motoneurons in models for ALS linked to a mutation in the gene coding for Cu,Zn superoxide dismutase (SOD1) and in ALS patients as well. Aggregation of mutant SOD1 in the cytoplasm and/or into mitochondria has been repeatedly proposed as a main culprit for the degeneration of motoneurons. It is, however, still debated whether SOD1 aggregates represent a cause, a correlate or a consequence of processes leading to cell death. We have exploited the ability of glutaredoxins (Grxs) to reduce mixed disulfides to protein thiols either in the cytoplasm and in the IMS (Grx1) or in the mitochondrial matrix (Grx2) as a tool for restoring a correct redox environment and preventing the aggregation of mutant SOD1. Here we show that the overexpression of Grx1 increases the solubility of mutant SOD1 in the cytosol but does not inhibit mitochondrial damage and apoptosis induced by mutant SOD1 in neuronal cells (SH-SY5Y) or in immortalized motoneurons (NSC-34). Conversely, the overexpression of Grx2 increases the solubility of mutant SOD1 in mitochondria, interferes with mitochondrial fragmentation by modifying the expression pattern of proteins involved in mitochondrial dynamics, preserves mitochondrial function and strongly protects neuronal cells from apoptosis. The toxicity of mutant SOD1, therefore, mostly arises from mitochondrial dysfunction and rescue of mitochondrial damage may represent a promising therapeutic strategy.

Related Reagents:
Parkin
Parkin-2
PARK2 Co-regulated (PACRG)
PARK7 (DJ-1)
LRRK2 (PARK8)
Neurodegenerative Disease Research Antibodies

Neurodegenerative Disease Research Proteins
Neurotransmission -Neurotransmission Research Antibody Categories
Neurotrophins and Growth Factor Antibodies
Neuron-Glial Expressed-Includes Neurotrophin Proteins

Apoptosis Research Reagents-Apoptosis Categories-includes: detection kits, antibodies and proteins

Primary Neurons and Astrocytes -Primary human, rat and mouse neurons and astrocytes by Category

TRHR1 and LepRb receptors and Thermogenesis

2010-09-15T17:44:00.000-07:00

I would like to thank Montina Van Meter, Lab Manager, Autonomic Neuroscience at Pennington Biomedical Research Center, for alerting me to this just published study. Included are excellent images of stained LepRb (OB-Rb) and GAD1 expressing neurons localized in loose clusters of cells in the DMN, NST, and the VLM.

This study focus on identifying loci in the hindbrain where leptin and TRH act synergistically to increase thermogenesis. Since thermogenic processes are at the root of how our bodies regulate energy, understanding the related expression and signaling pathways could be key to finding therapies for obesity.

Maria J. Barnes, Richard C. Rogers, Montina J. Van Meter and Gerlinda E. Hermann. Co-localization of TRHR1 and LepRb receptors on neurons in the hindbrain of the rat. doi:10.1016/j.brainres.2010.07.094

Example images: Distribution of LepRb+ fibers in hindbrain. LepRb-ir (red) fibers and varicosities are seen among TRHR1-ir (green) cells and fibers. These red and green fibers are adjacent and co-mingle but do not show co-localization of receptors. This pattern is seen in (A) fascicles of the solitary tract (ST); (B) raphe pallidus (RP), and (C) raphe obscurrus (RO). (D) Border between the medial solitary nucleus (NST) and the area postrema (AP; white dashed line) showing an abundance of LepRb-ir (red) fibers and
neurons (white arrows for selected neurons) in the NST but not the AP. (E) LepRb-ir staining is suppressed by pretreatment of tissue with LepRb epitope blocking peptide. (F) TRHR1-ir staining is suppressed by treatment with excess TRHR1. Scale bar A–D=100 microns; E, F=300 microns. cc=central canal.

Abstract: We have reported a highly cooperative interaction between leptin and thyrotropin releasing hormone (TRH) in the hindbrain to generate thermogenic responses (Hermann et al., 2006) (Rogers et al., 2009). Identifying the locus in the hindbrain where leptin and TRH act synergistically to increase thermogenesis will be necessary before we can determine the mechanism(s) by which this interaction occurs. Here, we performed heat-induced epitope recovery techniques and in situ hybridization to determine if neurons or afferent fibers in the hindbrain possess both TRH type 1 receptor and long-form leptin receptor [TRHR1; LepRb, respectively]. LepRb receptors were highly expressed in the solitary nucleus [NST], dorsal motor nucleus of the vagus [DMN] and catecholaminergic neurons of the ventrolateral medulla [VLM]. All neurons that contained LepRb also contained TRHR1. Fibers in the NST and the raphe pallidus [RP] and obscurrus [RO] that possess LepRb receptors were phenotypically identified as glutamatergic type 2 fibers (vglut2). Fibers in the NST and RP that possess TRHR1 receptors were phenotypically identified as serotonergic [i.e., immunopositive for the serotonin transporter; SERT]. Co-localization of LepRb and TRHR1 was not observed on individual fibers in the hindbrain but these two fiber types co-mingle in these nuclei. These anatomical arrangements may provide a basis for the synergy between leptin and TRH to increase thermogenesis.

Related Reagents:
Leptin and Leptin Receptor Antibodies

Leptin Proteins

Diabetes and Obesity Research

Antibodies

Diabetes and Obesity Proteins

Isolation of Medulloblastoma Stem Cells (Video Protocol)

2010-09-14T05:27:00.000-07:00

I am pleased to present this excellent video. It also has excellent images using key stem cell markers as the cells undergo differentiation:

JoVE: Isolation, Enrichment, and Maintenance of Medulloblastoma
...

Sep 1, 2010 ... GFAP antibody, Neuromics, CH22102,
Chicken, 1:1000. Tuj1 antibody, Sigma, T5076, Mouse, 1:2000. NeuN
antibody, Millipore, MAB377, Mouse, ...

www.jove.com

Potential Therapeutic Targets for Bone Cancer Pain-P2X Receptors

2010-09-09T15:14:00.000-07:00

Cancer pain is difficult to treat as it appears to be driven simultaneously by inflammatory, neuropathic and tumorigenic mechanisms. I have reported on multiple occasions publication referencing use of our Pain and Inflammation Research Antibodies in studying bone cancer pain.

I would like to alert you to the latest reference:

Timothy K. Y. Kaan, Ping K. Yip, Sital Patel, Meirion Davies, Fabien Marchand, Debra A. Cockayne, Philip A. Nunn, Anthony H. Dickenson, Anthony P. D. W. Ford, Yu Zhong, Marzia Malcangio, and Stephen B. McMahon Systemic blockade of P2X3 and P2X2/3 receptors attenuates bone cancer pain behaviour in rats. Brain, September 2010; 133: 2549 - 2564.

......Slides were then incubated with rabbit anti-P2X3 (1:2000, Neuromics) and sheep anti-calcitonin gene-related peptide (1:1000, Biomol...anti-beta-III-tubulin (1:4000, Promega) and guinea pig anti-P2X3 (1:100, Neuromics). The next day, after three washes with phosphate-buffered......

Summary: Pain remains an area of considerable unmet clinical need, and this is particularly true of pain associated with bone metastases, in part because existing analgesic drugs show only limited efficacy in many patients and in part because of the adverse side effects associated with these agents. An important issue is that the nature and roles of the algogens produced in bone that drive pain-signalling systems remain unknown. Here, we tested the hypothesis that adenosine triphosphate is one such key mediator through actions on P2X3 and P2X2/3 receptors, which are expressed selectively on primary afferent nocioceptors, including those innervating the bone. Using a well-established rat model of bone cancer pain, AF-353, a recently described potent and selective P2X3 and P2X2/3 receptor antagonist, was administered orally to rats and found to produce highly significant prevention and reversal of bone cancer pain behaviour. This attenuation occurred without apparent modification of the disease, since bone destruction induced by rat MRMT-1 carcinoma cells was not significantly altered by AF-353. Using in vivo electrophysiology, evidence for a central site of action was provided by dose-dependent reductions in electrical, mechanical and thermal stimuli-evoked dorsal horn neuronal hyperexcitability following direct AF-353 administration onto the spinal cord of bone cancer animals. A peripheral site of action was also suggested by studies on the extracellular release of adenosine triphosphate from MRMT-1 carcinoma cells. Moreover, elevated phosphorylated-extracellular signal-regulated kinase expression in dorsal root ganglion neurons, induced by co-cultured MRMT-1 carcinoma cells, was significantly reduced in the presence of AF-353. These data suggest that blockade of P2X3 and P2X2/3 receptors on both the peripheral and central terminals of nocioceptors contributes to analgesic efficacy in a model of bone cancer pain. Thus, systemic P2X3 and P2X2/3 receptor antagonists with central nervous system penetration may offer a promising therapeutic tool in treating bone cancer pain.

Related Reagents:

All Purinergic Receptors
Neurotransmission Research Antibodies

Angiogenesis Research Reagents

2010-08-08T08:03:00.000-07:00

The processes of angiogenesis and neurogenesis show striking similarities. Given our strong roots in providing Neuroscience research reagents, it is a natural extension for us to provide potent reagents for the studying the growth of new blood vessels.

Angiogensis processes are important for healing wounds and for restoring blood flow to tissues after injury or insult. In females, it also occurs during the monthly reproductive cycle and during pregnancy(to build the placental circulation between mother and fetus.

Images: Increased arteriogenesis and hyperemia in TSP2-null mice. Smooth muscle actin-positive and ephrin B2-positive vessels in WT (F, H) and TSP2-null (G, I). DOI: 10.2353/ajpath.2008.080128.

Angiogenesis-dependent diseases result when new blood vessels either grow excessively or insufficiently. These disease include: cancers, diabetic blindness, age-related macular degeneration, rheumatoid arthritis, psoriasis, neurodegeneration and many more.

Product Categories

Let-7 microRNAs and Nociceptive Pain

2010-07-29T07:51:00.000-07:00

Our Opioid Receptor Antibodies have set a potent standard for studying Nociceptive and Neuropathic Pain. Related Publications.

We want to recognize Dr. Zaijie Jim Wang and his team for being the first to use our Mu Opioid Receptor for studying the potential role of microRNAs in Nociception.

Ying He, Cheng Yang, Chelsea M. Kirkmire, and Zaijie Jim Wang. Regulation of Opioid Tolerance by let-7 Family MicroRNA Targeting the µ Opioid Receptor. The Journal of Neuroscience, July 28, 2010, 30(30):10251-10258; doi:10.1523/JNEUROSCI.2419-10.2010
Abstract: MicroRNA has emerged as a critical regulator of neuronal functions. This study aimed to test whether let-7 microRNAs can regulate the µ opioid receptor (MOR) and opioid tolerance. Employing bioinformatics, we identified a let-7 binding site in the 3'-untranslated region (UTR) of MOR mRNA, which was experimentally confirmed as a direct target of let-7. The repressive regulation of MOR by let-7 was revealed using a LNA-let-7 inhibitor to knockdown let-7 in SH-SY5Y cells. Conversely, morphine significantly upregulated let-7 expression in SH-SY5Y cells and in a mouse model of opioid tolerance. The LNA-let-7 inhibitor decreased brain let-7 levels and partially attenuated opioid antinociceptive tolerance in mice. Although chronic morphine treatment did not change overall MOR transcript, polysome-associated mRNA declined in a let-7-dependent manner. let-7 was identified as a mediator translocating and sequestering MOR mRNA to P-bodies, leading to translation repression. These results suggest that let-7 plays an integral role in opioid tolerance.

Western blot analysis. Western blot analysis was performed as previously described (Tang et al., 2006) using the anti-µ opioid receptor antibody (1:1000; Neuromics). The expression of β-actin was similarly determined from the same blots using a monoclonal antibody (1:10,000; Sigma).
For immunofluorescence analysis, the antibody for hDcp1a (Santa Cruz Biotechnology) and MOR were used at 1:500 and 1:5000 dilutions, respectively. Secondary anti-goat and anti-mouse antibodies labeled with Alexa 488 and Alexa 594 fluorochromes (Invitrogen), respectively, were used at 1:500 dilutions.

Related Reagent Links:
All Opioid Receptor Antibodies
Pain and Inflammation
Neurotransmission Research Antibodies-GPCRs, Ligand Gated Ion Channels,
Biogenic Amines and more
i-Fect Transfection Kit -gene silencing of DOR,
NaV1.8 tetrodotoxin-resistant sodium channel, NTS2 and more in-vitro and in vivo
Primary Neurons and Astrocytes-Primary human,
rat and mouse neurons and astrocytes

δ- and μ-opioid receptors co-expression and Nociceptive Pain

2010-07-26T07:14:00.000-07:00

Dr. Tomas Hokfelt and his team at Karolinska Institute recently published use of our Opioid Receptor Antibodies and Substance P Antibody.They show the interplay of DOR and MOR in modulation of nociceptive afferent transmission and opioid analgesia.

Hai-Bo Wanga, Bo Zhaoa, Yan-Qing Zhonga, Kai-Cheng Li, Zi-Yan Li, Qiong Wang, Yin-Jing Lua, Zhen-Ning Zhang, Shao-Qiu He, Han-Cheng Zheng, Sheng-Xi Wu, Tomas G. M. Hökfelt, Lan Baob, and Xu Zhanga. Coexpression of δ- and μ-opioid receptors in nociceptive sensory neurons. PNAS July 20, 2010 vol. 107 no. 29 13117-13122.

Immunostaining. Adult rats, mice, and Oprd1 exon 1-deleted mice were fixed. Cryostat sections of L4 and L5 DRGs and spinal cord segments were processed for immunofluorescence staining (13) with Rb anti-DOR13–17 (1:2,000–1:60,000; DiaSorin and 1:4,000–1:60,000; Neuromics), Rb anti-DOR12–18 (1:30,000–1:120,000; Alomone), Rb anti-DOR1358–372 (1:1,000–1:2,000; Lifespan Biosciences), Rb anti-MOR (1:1,000; Neuromics); guinea pig anti-SP (1:500; Neuromics), and mouse anti-CGRP (1:1,000; Biogenesis) antibodies. IB4-labeling was carried out with fluorescein-labeled GSL I-IB4 (1:200). The Myc-DOR1–transfected HEK293 cells and neurons were fixed and processed with mouse anti-Myc antibodies (1:500; DSHB). Nuclear DAPI staining was used to indicate HEK293 cells in control experiments.

Images: Distinct distribution patterns of DORs in subsets of DRG neurons of mice. Immunostaining with antibodies against DOR13–17 [A: 1:30,000, antibody 1 (ab #1); DiaSorin and C: antibody 2 (ab #2); Neuromics] shows DORs in small DRG neurons and afferent fibers in spinal laminae I–II. This immunostaining pattern is abolished by the antiserum preabsorption or the deletion of Oprd1 exon 1. Reduction in immunostaining is quantitatively assayed by determining the percentage of positive DRG neurons (B; n = 6) and fluorescence intensity (Ifluo.) in the laminae I–II (D; n = 5). **P < 0.01; ***P < 0.001. (Scale bars: A and C, 40 μm.). DOR labeling (anti-DOR13–17, 1:30,000; DiaSorin) associated with vesicles in peptidergic small DRG neurons (E and F) is absent in Oprd1 exon 1-deleted mice (G). Colocalization of DORs and neuropeptides is shown by correlated peaks of Ifluo. measured along lines. (Scale bar: 8 μm.) (H) Immunostaining with antibodies against DOR12–18 (1:60,000; Alomone) shows the presence of DORs on the cell surface of large DRG neurons of mice. (Scale bar: 25 μm.) This staining pattern is abolished by preabsorption and is absent in Oprd1 exon 1-deleted mice. (Scale bar: 80 μm.) (I) Triple-immunostaining shows that DOR+ large DRG neurons contain neither SP nor CGRP. (Scale bar: 80 μm.)

Immunoblotting.The samples were processed for SDS/PAGE, transferred, probed with Rb antibodies against MOR (1:500; Neuromics), phospho-DOR1 (1:1,000; Neuromics), phospho-MOR (1:1,000; Neuromics), Myc (1:500; DSHB), Flag (1:1,000; Sigma), or actin (1:50,000; Chemicon) and visualized with enhanced chemiluminescence (19).

ORL1-Whole Serum

All Opioid Receptor Antibodies

Pain and Inflammation

Neurotransmission Research Antibodies-GPCRs, Ligand Gated Ion
Channels, Biogenic Amines and more

i-Fect Transfection Kit -gene silencing
of DOR, NaV1.8 tetrodotoxin-resistant sodium channel, NTS2 and more
in-vitro and in vivo

Primary Neurons and Astrocytes-Primary
human, rat and mouse neurons and astrocytes

Staining Neuron-Glial Cultures-Related Markers

2010-07-19T13:15:00.000-07:00

I have been receiving a growing number of requests for best techniques related to staining cultures of primary neurons and glia. I wanted to share this short, step by step protocol.

These requests are often catalyzed by a search of our growing Neuron/Glial Markers catalog. The objective being to find the right markers for a particular assay. I wanted to share examples of the potency of several Neurofilament or NF markers for labeling neurons:

1. Neurofilament NF-L-Mouse Monoclonal Antibody (Clone: DA2) and Neurofilament alpha-internexin/NF66-Whole Serum-Rabbit Antibody

Images: Cells grown from adult rat brainLarge cell in middle is stained with mouse monoclonal to NF-L clone DA2 (green). Another type of neuronal lineage cell was stained with rabbit polyclonal to alpha-internexin (red). These cells were mitotic but had several characteristics of neurons. Rat spinal cord homogenate showing the major intermediate filament proteins of the nervous system (lane 1). The remaining lanes show blots of this material stainted with various antibodies including NF-L. Protocols on data-sheet.

2. Neurofilament NF-H, phosphylated-Mouse Monoclonal and Neurofilament NF-L-Purified Chicken Polyclonal.

Image: View of mixed neuron/glial cultures stained with chicken polyclonal NF-L (red) and phosphorylated NF-H The NF-L protein is assembled into neurofilaments which are found throughout the axons, dendrites and perikarya of these cells. In contrast the phosphorylated NF-H has a much rmore restricted expression pattern, being found only in developed axonal neurofilaments. Since both proteins are found in neurofilaments, the red and green patterns overlap, so that neurofilaments containing NF-L and phosphorylated NF-H appear yellowish. In contrast neurofilaments containing only NF-L appear red. Protocol on datasheet.

Neurofilament Markers

TRPV1 & P2X3-Daily Double

2010-07-06T11:46:00.000-07:00

Low pH and Chronic Muscle Pain

Our Pain and Inflammation Antibodies are routinely used for chronic pain. I would like to highlight a recent publication referencing use of our Guinea Pig TRPV1 and Pig 2X3 Antibodies and Blocking Peptides:

James P. Lund,Somayeh Sadeghi,Tuija Athanassiadis, Nadia Caram Salas, François Auclair, Benoît Thivierge, Isabel Arsenault, Pierre Rompré, Karl-Gunnar Westberg, and Arlette Kolta.Assessment of the Potential Role of Muscle Spindle Mechanoreceptor Afferents in Chronic Muscle Pain in the Rat Masseter Muscle. PLoS One. 2010; 5(6): e11131. Published online 2010 June 15 doi:10.1371/journal.pone.0011131.

Conclusions/Significance

Low pH leads to changes in several electrical properties of MSA, including initiation of ectopic action potentials which could propagate centrally but could also invade the peripheral endings causing glutamate release and activation of nearby nociceptors within the spindle capsule. This peripheral drive could contribute both to the transition to, and maintenance of, persistent muscle pain as seen in some “functional” pain syndromes.

Images: Photomicrographs of trigeminal ganglion neurons stained with TRPV1 and P2X3.

Related Reagents:

Pain and Inflammation Research Antibodies

Neurotransmission

Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes

Otx2 (Orthodenticle Homeobox 2) and Parkinson's Disease

2010-06-28T04:37:00.000-07:00

Our Otx2 Antibody is a potent marker for Human, Mouse and Rat Midbrain Dopamanergic Progenitors.

This is confirmed by a recent publication by Dr. Ole Isaacson et al:

Chee Yeun Chung, Pawel Licznerski, Kambiz N. Alavian, Antonio Simeone, Zhicheng Lin, Eden Martin, Jeffery Vance and Ole Isacson. The transcription factor orthodenticle homeobox 2 influences axonal projections and vulnerability of midbrain dopaminergic neurons. Brain Advance Access published online on June 23, 2010 Brain, doi:10.1093/brain/awq142... anti-Otx2 (Neuromics, 1:500)...

Abstract: Two adjacent groups of midbrain dopaminergic neurons, A9 (substantia nigra pars compacta) and A10 (ventral tegmental area), have distinct projections and exhibit differential vulnerability in Parkinson’s disease. Little is known about transcription factors that influence midbrain dopaminergic subgroup phenotypes or their potential role in disease. Here, we demonstrate elevated expression of the transcription factor orthodenticle homeobox 2 in A10 dopaminergic neurons of embryonic and adult mouse, primate and human midbrain. Overexpression of orthodenticle homeobox 2 using lentivirus increased levels of known A10 elevated genes, including neuropilin 1, neuropilin 2, slit2 and adenylyl cyclase-activating peptide in both MN9D cells and ventral mesencephalic cultures, whereas knockdown of endogenous orthodenticle homeobox 2 levels via short hairpin RNA reduced expression of these genes in ventral mesencephalic cultures. Lack of orthodenticle homeobox 2 in the ventral mesencephalon of orthodenticle homeobox 2 conditional knockout mice caused a reduction of midbrain dopaminergic neurons and selective loss of A10 dopaminergic projections. Orthodenticle homeobox 2 overexpression protected dopaminergic neurons in ventral mesencephalic cultures from Parkinson’s disease-relevant toxin, 1-methyl-4-phenylpyridinium, whereas downregulation of orthodenticle homeobox 2 using short hairpin RNA increased their susceptibility. These results show that orthodenticle homeobox 2 is important for establishing subgroup phenotypes of post-mitotic midbrain dopaminergic neurons and may alter neuronal vulnerability.

Image: Characterization of the human neuroectodermal precursors. Otx2 Staining of forebrain-midbrain rosettes (dilution 1:1000).

All Publications Referencing Neuromics Purified Goat Polyclonal Otx2

Related Reagents to Consider:

Nestin as a Marker for Astrocytomas

2010-06-25T04:14:00.000-07:00

I recently highlighted the growing parade of pubs referencing use of our reagents for Cancer Research.

I would like to add a new one. Angogenesis of Astrocytomas show stem like properties. This makes our Nestin Antibodies excellent markers.

J H Tchaicha, A K Mobley, M G Hossain, K D Aldape and J H McCarty. A mosaic mouse model of astrocytoma identifies αvβ8 integrin as a negative regulator of tumor angiogenesis. Oncogene , (7 June 2010) doi:10.1038/onc.2010.199...chicken anti-Nestin IgY (Neuromics, Edina, MN, USA)...

Abstract: Angiogenesis involves a complex set of cell–cell and cell–extracellular matrix (ECM) interactions that coordinately promote and inhibit blood vessel growth and sprouting. Although many factors that promote angiogenesis have been characterized, the identities and mechanisms of action of endogenous inhibitors of angiogenesis remain unclear. Furthermore, little is known about how cancer cells selectively circumvent the actions of these inhibitors to promote pathological angiogenesis, a requisite event for tumor progression. Using mosaic mouse models of the malignant brain cancer, astrocytoma, we report that tumor cells induce pathological angiogenesis by suppressing expression of the ECM protein receptor αvβ8 integrin. Diminished integrin expression in astrocytoma cells leads to reduced activation of latent TGFβs, resulting in impaired TGFβ receptor signaling in tumor-associated endothelial cells. These data reveal that astrocytoma cells manipulate their angiogenic balance by selectively suppressing αvβ8 integrin expression and function. Finally, these results show that an adhesion and signaling axis normally involved in developmental brain angiogenesis is pathologically exploited in adult brain tumors.

Related Reagents:

Nestin Mouse Monoclonal-Cat#:MO15012
Nestin-Mouse Monoclonal-Cat#:MO15056
Nestin-Goat Polyclonal
Stem Cell Research Reagents

Cancer Reagents Pubs-Capabilities Update

2010-06-11T11:20:00.000-07:00

We continue to grow our capaibilities and abilities to serve Cancer Researchers.

We recently highlighted the potency of our i-Fect ™ siRNA transfection kits for deliveriny siRNA to glioblastomas.

Joseph George, Naren L. Banik, Swapan K. Ray. Combination of hTERT Knockdown and IFN-γ Treatment Inhibited Angiogenesis and Tumor Progression in Glioblastoma. Clin Cancer Res 2009;15(23):7186–95

...with i-Fect transfection reagent (Neuromics) to obtain 5 μg DNA/10 μL of injection volume...

Here're several new publications highlighting use of our Cancer Research Antibodies:

Mauricio P. Pinto, Melanie M. Badtke, Michelle L. Dudevoir, J. Chuck Harrell, Britta M. Jacobsen and Kathryn B. Horwitz. Vascular Endothelial Growth Factor Secreted by Activated Stroma Enhances Angiogenesis and Hormone-Independent Growth of Estrogen Receptor–Positive Breast Cancer. Cancer Research 70, 2655, April 1, 2010. Published Online First March 23, 2010; doi: 10.1158/0008-5472.CAN-09-4373 © 2010 American Association for Cancer Research.

...Phosphorylated extracellular signal-regulated kinase (p-ERK) was assayed by immunohistochemistry (rabbit polyclonal; Neuromics). Statistical analyses Data were analyzed with GraphPad software using either Student's t test or ANOVA followed by a Tukey's...

Nina Bergelin, Christoffer Löf, Sonja Balthasar, Veronica Kalhori, and Kid Törnquist. S1P1, and VEGFR-2 Form a Signaling Complex with Extracellularly Regulated Kinase 1/2 and Protein Kinase C-alpha Regulating ML-1 Thyroid Carcinoma Cell Migration. This version published online on May 25, 2010. Endocrinology, doi:10.1210/en.2009-1387

...conjugated goat antirabbit from Bio-Rad Laboratories (Hercules, CA). Rearranged in transformation (RET) antibody was from Neuromics (Edina, MN). Secondary antibodies (Alexa Fluor goat antirabbit 568 and goat antimouse 488) for immunocytochemistry were obtained from MolecularProbes...

New Markers:

Ogg1, Biotinylated
Ogg1, HRP Conjugated
p95/NBS1
Pin-1

Image: HeLa cells stained with Pin-1 (1:1,000 dilution, green) and fibrillarin (red). Pin-1 stains the nuclear matrix and, much more faintly, the cytoplasm. The fibrillarin antibody marks nucleoli.

STEMEZ(TM) hNP1 Cells and Neuroprotection Studies

2010-06-06T04:33:00.000-07:00

Neuroinflammation has been shown to be a potent negative regulator of stem cell and progenitor cell proliferation in the neurogenic regions of the brain.

Here researchers used our STEMEZ(TM) hNP1 Human Neural Progenitors Expansion Kit to study the neuroprotection capabilities of a propriety nutraceutical formulation.

Adam D. Bachstetter, Jennifer Jernberg, Andrea Schlunk, Jennifer L. Vila, Charles Hudson, Michael J. Cole, R. Douglas Shytle, Jun Tan, Paul R. Sanberg, Cyndy D. Sanberg, Cesario Borlongan, Yuji Kaneko, Naoki Tajiri, Carmelina Gemma, Paula C. Bickford. Spirulina Promotes Stem Cell Genesis and Protects against LPS Induced Declines in Neural Stem Cell Proliferation. PLoS ONE 5(5): e10496. doi:10.1371/journal.pone.0010496.

Abstract:Adult stem cells are present in many tissues including, skin, muscle, adipose, bone marrow, and in the brain. Neuroinflammation has been shown to be a potent negative regulator of stem cell and progenitor cell proliferation in the neurogenic regions of the brain. Recently we demonstrated that decreasing a key neuroinflammatory cytokine IL-1β in the hippocampus of aged rats reversed the age-related cognitive decline and increased neurogenesis in the age rats. We also have found that nutraceuticals have the potential to reduce neuroinflammation, and decrease oxidative stress. The objectives of this study were to determine if spirulina could protect the proliferative potential of hippocampal neural progenitor cells from an acute systemic inflammatory insult of lipopolysaccharide (LPS). To this end, young rats were fed for 30 days a control diet or a diet supplemented with 0.1% spirulina. On day 28 the rats were given a single i.p. injection of LPS (1 mg/kg). The following day the rats were injected with BrdU (50 mg/kg b.i.d. i.p.) and were sacrificed 24 hours after the first injection of BrdU. Quantification of the BrdU positive cells in the subgranular zone of the dentate gyrus demonstrated a decrease in proliferation of the stem/progenitor cells in the hippocampus as a result of the LPS insult. Furthermore, the diet supplemented with spirulina was able to negate the LPS induced decrease in stem/progenitor cell proliferation. In a second set of studies we examined the effects of spirulina either alone or in combination with a proprietary formulation (NT-020) of blueberry, green tea, vitamin D3 and carnosine on the function of bone marrow and CD34+ cells in vitro. Spirulina had small effects on its own and more than additive effects in combination with NT-020 to promote mitochondrial respiration and/or proliferation of these cells in culture. When examined on neural stem cells in culture spirulina increased proliferation at baseline and protected against the negative influence of TNFα to reduce neural stem cell proliferation. These results support the hypothesis that a diet enriched with spirulina and other nutraceuticals may help protect the stem/progenitor cells from insults. Figure 7. Spirulina increases proliferation of human neural stem cells in vitro and protects against a TNFα insult.

Figure: Human neural progenitors grown under proliferation conditions were assessed by MTT assay (A) or BrdU (B) for the effects of spirulina (125 ng/ml) or NT-020 (500 ng/ml) or the two treatments combined in the presence or absence of TNFα (20 ng/ml) for 72 hours. (A) The MTT assay shows that spirulina alone or NT-020 alone increase proliferation; surprising, the in combination proliferation is decrease compare to control ** p less tan 0.005.

Primary Neurons and Astrocytes-Primary
human, rat and mouse neurons and astrocytes

Neuron/Glial Marker Antibodies

Neurotrophins and Growth Factor
Antibodies

Stem Cell Research Reagents-includes
cells, antibodies, proteins, media and FACS kits.

STEMEZ(TM) hNP1 Human Neural Progenitors and hN2 Primary Neurons Differentiation and Expression

2010-05-29T13:48:00.000-07:00

I have received a growing number of requests regarding differentiation and expression patterns of our STEMEZ^TM cells. This is intended help you gain a clearer understanding. The focus of this document is G-protein Coupled Receptor Expression Patterns. This information was presented by Dr. Steve Stice and his team and the 2008 Neuroscience Conference.

INTRODUCTION

Human embryonic stem cells and their progeny can provide a novel Distribution of detectable transcripts for three cell populations tissue source for understanding developmental pathways, pharmaceutical screening and tissue replacement therapies. G-protein coupled receptors(GPCRs) comprise the largest cell-surface receptor superfamily and are the largest class of drug targets. The study of GPCR signaling in hES cells allows signaling mechanisms to be studied in endogenously expressed receptors in non-transformed cells. We characterized GPCR transcript expression in three cellular populations at different developmental stages: WAO9 human embryonic stem cells, Wa09 derived STEMEZ hNP1 and differentiated hN2 cells maintained 1 week in culture.

Goal: To characterize GPCR transcript expression in human hES cell derived neural tissue

CONCLUSIONS
• hES cells displayed the widest array of GPCR transcripts, while neural progenitors displayed the most restricted population.
• The Frizzled (FZD) family of receptors were among the most abundantly expressed transcripts across all populations.
• Neural progentitors up-regulated GPCR transcripts important to brain angiogenesis, cell proliferation, neurogenesis and cell adhesion.
• Further differentiated hN2 cells displayed up-regulation of a wider population of transcripts including GPCRs involved with neurotransmission.
• Functional assays demonstrated responses to sphingosine-1-phosphate in both hNP1 and hN2 populations of cells.
• hES cells and their derived tissue provide a unique model to study endogenous GPCR signaling in non-transformed cells for drug screening applications and to further our understanding of GPCRs role in developmental pathways.

G-protein Coupled Receptor Expression Patterns Are Altered as Human Embryonic Stem Details(pdf - 367Kb). From Poster Presented at Neuroscience 2008 by Dr. Steve Stice et al.

Dr. Steve Stice to Present the Power of StemEZ Neural Cells

2010-05-25T17:09:00.001-07:00

Dr. Steve Stice to Present the Power of StemEZ Neural Cells

Posted using ShareThis

I have profiled Steve Stice's research here. The focus has been the excellent research results he and his team at ArunA Biomedical have generated with STEMEZ(TM) hN2 Human Neurons and hNP1 Human Neural Progenitors.

The story continues. He will be presenting the latest at the 9th Annual World Pharmaceutical Congress in Philadelphia, June 14. Topics include: using these neural cell lines to study neurotoxicity in cell-based assays and disease modeling. Recent work conducted in outside laboratories demonstrates that these lines are more sensitive to environmental toxicants than traditional cellular models.

Sample high throughput assay applications:

Cell morphology and neurite outgrowth

Cell signaling and transcription factor expression

Receptor and ion channel function

Cytotoxicity
Apoptosis, genotoxicity and DNA damage

These capabilities has been confirmed by our customers. I look for the use of the STEMEZ cell lines to continue to grow as researchers discover their value in Drug Discovery and Basic Neuroscience capabilities.

Amp up Your Results!

2010-05-21T06:58:00.001-07:00

Labeling kits, tags, secondary antibodies and related reagents

Posted using ShareThis

Labeling and tagging is an important step in your research process. This often drives the wow factor in published results. We offer some of the best and brightest including:

CHROMEO^TMsity-exhibit superior luminescence properties,
including a broad range of fluorescence excitation and emission, large
Stokes shifts, limited photobleaching and a broad pH tolerance.

ELISA Buffers and Diluents

Solulink™ Labeling Kits and Beads-The most efficient labeling kits delivering ready-to-use conjugates for the novice or the expert!

Cytoplasmic and Nuclear Staining

Strep-Tag®-One-STrEP-tag for protein complex purification.

Image: CHROMEOsity 488: HeLa cells were stained with alpha Tubulin mouse mAb (Clone 5-B-1-2) and Chromeo 488 Goat anti-mouse IgG. The nuclei have been counterstained with DAPI.

Apoptosis Signaling-Visualization and Measurement.

2010-05-16T09:27:00.000-07:00

Apoptosis-Oxidative Stress Research Reagents are widely used and frequently referenced in customer publications. We work hard to keep our fingers on the pulse of how they are utilized across the many research areas important to our customers and add new reagents based on evolving requirements.

I recently posted publications referencing our MitoPT^TM Kits for quantitating Tumor Apoptosis

New Pub referencing Polycaspase Assay Kit, green: L. Wei, D. Ding and R. Salvi. Salicylate-induced degeneration of cochlea spiral ganglion neurons-apoptosis signaling.
doi:10.1016/j.neuroscience.2010.03.015.

Images: Typical confocal photomicrographs of SGN stained with Polycaspase Assay Kit (green) and with an antibody against neuronal III ß-tubulin (red) to identify SGN. (A) In control cultures, most SGN have large, oval shaped soma and neurites extending from the soma; note absence of polycaspase labeling (green). (B) SGN treated for 3 h with 5 mM SS; polycaspase labeling was present on SGN with shrunken soma. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

Related Reagents:

Polycaspase Assay Kit, green

Magic Red™ Real Time! Kits -Measure apoptosis in
whole living, intact cells - no lysis required

FLIVO™ Polycaspase Live!, in vivo Apoptosis Kits-New
-Designed for cancer and neurodegenerative disease applications.

FLICA™ in vitro Caspase Kits -Fast!-Use Caspase
kits to quantitate apoptosis via active caspases in whole, living
cells. These kits do not use ELISA or any antibodies for detection

FLISP™ Serine Protease Detection Kits -Measure
chymotrypsin-like protease activation in whole living cells.

MitoPT™ Kits -Quantitate mitochondrial
functionality and apoptosis

Angiogensis and CD antigens

2010-05-15T11:26:00.001-07:00

We consider everything genesis as a core focus area. This includes embryogenesis, angiogenesis, neurogenesis and stem cell expansion and differentiation. This is important because there is an intersection between reagents and methods for studying embryo development and genesis pathways in "adult systems".

CD antigens serve as markers for growth and development. We are always on the look out for Customer Publications that reference use of these reagents in related applications. Here's one that just pinged our radar.

Karim Harhouri, Abdeldjalil Kebir, Benjamin Guillet, Alexandrine Foucault-Bertaud, Serge Voytenko, Marie-Dominique Piercecchi-Marti, Caroline Berenguer, Edouard Lamy, Frédéric Vely, Pascale Pisano, L'Houcine Ouafik, Florence Sabatier, José Sampol, Nathalie Bardin, Françoise Dignat-George, and Marcel Blot-Chabaud Soluble CD146 displays angiogenic properties and promotes neovascularization in experimental hind-limb ischemia. Blood, May 2010; 115: 3843 - 3851
Anti-rat antibodies used in this study are: anti-CD117 (Neuromics) and anti-CD146

Abstract: CD146, an endothelial molecule involved in permeability and monocyte transmigration, has recently been reported to promote vessel growth. As CD146 is also detectable as a soluble form (sCD146), we hypothesized that sCD146 could stimulate angiogenesis. Experiments of Matrigel plugs in vivo showed that sCD146 displayed chemotactic activity on endogenous endothelial cells, and exogenously injected late endothelial progenitor cells (EPCs). Recruited endothelial cells participated in formation of vascular-like structures. In vitro, sCD146 enhanced angiogenic properties of EPCs, with an increased cell migration, proliferation, and capacity to establish capillary-like structures. Effects were additive with those of vascular endothelial growth factor (VEGF), and sCD146 enhanced VEGFR2 expression and VEGF secretion. Consistent with a proangiogenic role, gene expression profiling of sCD146-stimulated EPCs revealed an up-regulation of endothelial nitric oxide synthase, urokinase plasminogen activator, matrix metalloproteinase 2, and VEGFR2. Silencing membrane-bound CD146 inhibited responses. The potential therapeutic interest of sCD146 was tested in a model of hind limb ischemia. Local injections of sCD146 significantly reduced auto-amputation, tissue necrosis, fibrosis, inflammation, and increased blood flow. Together, these findings establish that sCD146 displays chemotactic and angiogenic properties and promotes efficient neovascularization in vivo. Recombinant human sCD146 might thus support novel strategies for therapeutic angiogenesis in ischemic diseases

Image: c-KIT staining of rat skin (epidermis). c-KIT detection was done using anti-rabbit Cy3 conjugated antibodies (red color). DAPI was used to counterstain cell nuclei (blue color).Working dilution: 1:100-1:300.

Related Reagents:

Immune Response Antibdodies

Immune Response Proteins

Cancer Research Antibodies

Cancer Research Proteins

Pain and Inflammation Research Antibodies

Neurodegenerative Disease Antibodies

Neurodegenerative Disease Proteins

Apoptosis Research Reagents

Stem Cell Research Reagents

TRPV1 Expression and Temporal Lobe Epilepsy

2010-05-08T12:33:00.000-07:00

Dr Bret. Smith and his lab at Tulane have demontrated a link between Temporal Lobe Epilepsy (TLE) and VR1 N-Terminus (TRPV1) expression.

In this excellent study they showed an increase in expression in TLE mice vs controls.

Muthu D. Bhaskaran and Bret N. Smith. Effects of TRPV1 activation on synaptic excitation in the dentate gyrus of a mouse model of temporal lobe epilepsy. doi:10.1016/j.expneurol.2010.01.021
...with polyclonal VR1 N-terminus (1:2500) (Neuromics, Edina)..

Abstract: Temporal lobe epilepsy (TLE) is a condition characterized by an imbalance between excitation and inhibition in the temporal lobe. Hallmarks of this change are axon sprouting and accompanying synaptic reorganization in the temporal lobe. Synthetic and endogenous cannabinoids have variable therapeutic potential in treating intractable temporal lobe epilepsy, in part because cannabinoid ligands can bind multiple receptor types. This study utilized in vitro electrophysiological methods to examine the effect of transient receptor potential vanilloid type 1 (TRPV1) activation in dentate gyrus granule cells in a murine model of TLE. Capsaicin, a selective TRPV1 agonist had no measurable effect on overall synaptic input to granule cells in control animals, but significantly enhanced spontaneous and miniature EPSC frequency in mice with TLE. Exogenous application of anandamide, an endogenous cannabinoid that acts at both TRPV1 and cannabinoid type 1 receptors (CB1R), also enhanced glutamate release in the presence of a CB1R antagonist. Anandamide reduced the EPSC frequency when TRPV1 were blocked with capsazepine. Western blot analysis of TRPV1 receptor indicated protein expression was significantly greater in the dentate gyrus of mice with TLE compared with control mice. This study indicates that a prominent cannabinoid agonist can increase excitatory circuit activity in the synaptically reorganized dentate gyrus of mice with TLE by activating TRPV1 receptors, and suggests caution in designing anticonvulsant therapy based on modulating the endocannabinoid system.

Images: Western blot detection of TRPV1 receptor expression in the dentate gyrus. A. Diagram of dentate gyrus showing the microdissected area (box). B. Western blot showing TRPV1 receptor expression in two untreated mice and in two pilocarpine-treated mice that survived SE. Actin was used as the loading control which did not change significantly. C. Graph showing a significant (p less than 0.05; n=4) TRPV1 expression in epileptic mice.

Related Reagents:

TRPV (Vanilloid); TRPM; TRPA and TRPCs

Neurotransmission Research Antibodies-GPCRs, Ligand Gated Ion Channels, Biogenic Amines and more

Primary Neurons and Astrocytes- Primary human, rat and mouse neurons and astrocytes by Category.

MitoPT for Studying Tumor Apoptosis

2010-04-24T07:27:00.000-07:00

We value our partnership with ICT. They provide our customers with potent and research proven Apoptosis Kits and Methods. Here we feature publications referencing our MitoPT™ Kits. These Kits easily assess changes in mitochondrial membrane potential. Changes in mitochondrial membrane potential can correlate with cytochrome c release and the initiation of apoptosis.
A431 cells, treated with predetermined IC50
concentration of novel anticancer agents, fluoresce green and orange-red with MitoPT JC-1. Data courtesy of Zayas/ Carro, Universidad Metropolitana.
Anticancer Effects of Alpinia pricei Hayata Roots.
CL Hsu, YS Yu, GC Yen. J. Agric. Food Chem., Jan 2010, 58 (4), pp 2201–2208.

Anticancer Effects of Flavonoid Derivatives Isolated from Millettia
reticulata Benth in SK-Hep-1 Human Hepatocellular Carcinoma Cells.
SC Fang, CL Hsu, HT Lin, GC Yen. J. Agric. Food Chem., Jan 2010, 58
(2), pp 814–820.

Mechanisms of Apoptotic Effects Induced by Resveratrol,
Dibenzoylmethane, and Their Analogues on Human Lung Carcinoma Cells.
CJ Weng, YT Yang, CT Ho, GC Yen. J. Agric. Food Chem., Jun 2009; 57
(12), pp 5235–5243.

CFR1, 5-HT2AR and Anxiety Behavior

2010-04-20T11:26:00.000-07:00

We have a potent offering of 5HT-Serotonin Antibodies. This is confirmed by our growing parade of customer publications referencing their use.

We are pleased to present a new publication referencing use of our 5HT (Serotonin) 2A Receptor Antibody. Dr. Stephen S G Ferguson and team have discovered a link between CFR1 and 5-HT2A Receptor expression:

Ana C Magalhaes,Kevin D Holmes,Lianne B Dale,Laetitia Comps-Agrar,Dennis Lee,Prem N Yadav, Linsay Drysdale, Michael O Poulter, Bryan L Roth, Jean-Philippe Pin, Hymie Anisman& Stephen S G Ferguson. CRF receptor 1 regulates anxiety behavior via sensitization of 5-HT2 receptor signaling. Nature Neuroscience. doi:10.1038/nn.2529. Published online11 April 2010.

Abstract: Stress and anxiety disorders are risk factors for depression and these behaviors are modulated by corticotrophin-releasing factor receptor 1 (CRFR1) and serotonin receptor (5-HT2R). However, the potential behavioral and cellular interaction between these two receptors is unclear. We found that pre-administration of corticotrophin-releasing factor (CRF) into the prefrontal cortex of mice enhanced 5-HT2R–mediated anxiety behaviors in response to 2,5-dimethoxy-4-iodoamphetamine. In both heterologous cell cultures and mouse cortical neurons, activation of CRFR1 also enhanced 5-HT2 receptor–mediated inositol phosphate formation. CRFR1-mediated increases in 5-HT2R signaling were dependent on receptor internalization and receptor recycling via rapid recycling endosomes, resulting in increased expression of 5-HT2R on the cell surface. Sensitization of 5-HT2R signaling by CRFR1 required intact PDZ domain–binding motifs at the end of the C-terminal tails of both receptor types. These data suggest a mechanism by which CRF, a peptide known to be released by stress, enhances anxiety-related behavior via sensitization of 5-HT2R signaling.

Images: (a) Dose response curves for 5-HT–stimulated inositol phosphate formation in HEK 293 cells transfected with FLAG–5-HT2AR and HA-CRFR1 and pretreated with or without 500 nM CRF for 30 min in the presence of dominant-negative dynamin I-K44A. The dose response curves represent the mean ± s.e.m. for four independent experiments. (b,c) Representative laser-scanning confocal micrographs showing the distribution of FLAG-5-HT2AR and HA-CRFR1 (b) and FLAG-5-HT2CR and HA-CRFR1 (c) in HEK 293 cells labeled with FLAG and HA antibodies at 4 °C and then warmed to 37 °C for 30 min in the absence of agonist. (d) Representative laser-scanning confocal micrographs showing the distribution of FLAG–5-HT2AR and HA-CRFR1 labeled with FLAG and HA antibodies at 4 °C and warmed to 37 °C for 30 min in the absence of agonist. (e) Representative laser-scanning confocal micrographs showing the distribution of FLAG–5-HT2AR and HA-CRFR1 transfected into rat cortical neurons labeled with FLAG and HA antibodies at 4 °C and treated with 500 nM CRF and warmed to 37 °C for 30 min. (f) Representative laser-scanning confocal micrographs showing the distribution of FLAG–5-HT2AR and HA-β2AR transfected into HEK 293 cells labeled with FLAG and HA antibodies at 4 °C and treated with 100 μM isoproterenol and warmed to 37 °C for 30 min. Micrographs are representative images of multiple cells imaged on three independent occasions. Scale bars represent 10 μm.

Related Reagents:
All 5HT-Serotonin Antibodies
Neurotransmission Research Antiboodies
Primary Neurons and Astrocytes
-Primary human, rat and mouse neurons and astrocytes

More on Neuromics' Neuron Markers

2010-04-13T05:17:00.000-07:00

I have multiple posts on the potency of our Neuron Markers. I am pleased to present yet another reference. This on features use of our Chicken Tyrosine Hydroxylase-TH antibody. It features staining of juxtaglomerular cells in the olfactory bulb of mice:

Hans-Ulrich Fried, U. Benjamin Kaupp and Frank Müller. Hyperpolarization-activated and cyclic nucleotide-gated channels are differentially expressed in juxtaglomerular cells in the olfactory bulb of mice. Cell Tissue Res. 2010 March; 339(3): 463–479. Published online 2010 February 6. doi: 10.1007/s00441-009-0904-9.

Image: TH antibody staining in ET-like cell populations within the Glomerulari (GL). Dilution 1:500

Related Reagents:

TH-Mouse Monoclonal Catalog#: MO20001
TH-Monoclonal-High Titer Catalog#:MO22941
All Neuron-Glial Markers
Neurodegenerative Disease Antibodies
Stem Cell Research Reagents
Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and astrocytes

Potent Neuron-Glial Markers

2010-03-26T12:16:00.000-07:00

We are recognized for having top shelf Neuron/Glial Marker Antibodies. We have an extensive catalog and have customer referencing use of these in a variety of applications, species and cell types.

Cell types include neural progenitors, neurons, glia, astrocytes, schwann cells and more. We are pleased to provide present a new publication referencing use of our MAP2 (Microtubule assoc. protein 2) Antibody for immunostaining of E17 primary mouse astrocytes.

Shelley Jacobs and Laurie C. Doering. Astrocytes Prevent Abnormal Neuronal Development in the Fragile X Mouse. J. Neurosci., Mar 2010; 30: 4508 - 4514 ; doi:10.1523/JNEUROSCI.5027-09.2010.

After 7 d in vitro (DIV), the cells were fixed with ice-cold (–20°C) methanol and processed for immunocytochemistry. After the appropriate serum block, the cells were incubated with primary antibodies overnight at 4°C. Secondary antibodies were applied for 3 h at room temperature. The following antibody, diluted in 1% BSA, was used: chicken microtubule-associated protein 2 (MAP2) (1:20,000; Neuromics) and anti-chicken FITC (1:100; Jackson ImmunoResearch Laboratories). Coverslips were mounted with Vectashield fluorescent mounting medium with 4`,6-diamidino-2-phenylindole (DAPI).

Images: Effects of astrocytes on the growth of hippocampal neurons in coculture at 7 DIV. E17 primary hippocampal neurons were cocultured with P0–P1 primary cortical astrocytes for 7 DIV in each of four coculture conditions. a, Immunofluorescent images of neurons in each of the four culture combinations. Neurons are stained with an antibody directed against the neuronal dendritic marker, MAP2. Scale bar, 100 µm. b, Quantification of percentage of surviving neurons at 7 DIV in each of the four culture conditions. Data shown are mean values ± SEM from two or three independent experiments (10–15 regions of 1.5 mm2 from 2 coverslips per experiment). Significant differences revealed by post hoc Tukey's tests are indicated (p less than 0.001).

Related Reagents

Stem Cell Research Antibodies

Stem Cell Research Reagents

Primary Neurons and Astrocytes-Primary
human, rat and mouse neurons and astrocytes

Primary Hypothalamic Neurons Cultures

2010-03-23T16:28:00.000-07:00

Our customers have had great success dissociating and culturing our Fresh E18 and E20Rat Primary Neuronal Tissue. They have proven useful for a variety of cell based assays.

Here're several publications referencing use of the neurons:

Lisette T. Arnaud, Natura Myeku and Maria E. Figueiredo-Pereira. Proteasome–caspase–cathepsin sequence leading to tau pathology induced by prostaglandin J2 in neuronal cells. Journal of Neurochemistry. Volume 110 Issue 1, Pages 328 - 342 .

Karunya K. Kandimalla1, Olenych G. Scott, Smita Fulzele1, Michael W. Davidson, Joseph F. Poduslo. Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid β Protein. PLoS ONE 4(2): e4627. doi:10.1371/journal.pone.0004627.

The most challenging of our neurons to culture are from the microdissect E18 rat Hypothalamus. Big Kudos to Dr. Dan Ryder for his excellent job culturing them. He also generously shared these images: (A) 5X1 (B) 40X5 Brightfield.

New Human Mesenchymal Stem Cells

2010-03-18T07:57:00.000-07:00

Neuromics' is responding to growing demand for Stem Cell Research Reagents.

This includes the addition of human stem cells giving researchers the ability to create consistent cultures of primary cells. Our first offering was STEMEZ ^TM hNP1 Human Neural Progenitors. We have received positive feedback on these regarding ease of use and quality.

We are pleased to announce the addition of Human Mesenchymal Stem Cells.

Image: Pancreas-derived human mesenchymal stem cells labeled with a CD44 monoclonal antibody conjugated to fluorescein isothiocyanate (FITC), which detects the CD44 cell surface protein.

They are isolated from human adult pancreas and can be induced to differentiate into beta-cells, which is a significant product in the diabetes research area. MSCs to Beta Cells Protocol.

This is extremely important due to the low yield of islets from patients/donors. These cells could be a functional equivalent to normal beta-cells and could restore proper endocrine function to the pancreas.

In addition, protocols are available to differentiate these cells into osteocytes, adipocytes, chondrocytes and hepatocytes.

We will keep you posted on customer results with this important new product.