Human Embryonic Stem Cell Differentiation Protocol

Here's a protocol that could be of interest to hESC researchers:

Elizabeth S Ng, Richard Davis, Edouard G Stanley & Andrew G Elefanty. A protocol describing the use of a recombinant protein-based, animal product-free medium (APEL) for human embryonic stem cell differentiation as spin embryoid bodies. Nature Protocols 3, - 768 - 776 (2008).

PDGFs in Non-small Cell Lung Cancer Tumor and Stromal Cells

Serving Cancer and Tumorigenesis Researhers is a central component to our business strategy. This means constantly building our catalog of Cancer Antibodies and Cancer Proteins.

I personally follow up with every customer that purchases our Cancer Research Reagents. This ensures they work as expected. In most cases, this is validate. If not, we fix the expressed issues.

We are now seeing more confirmation by the reagents being referenced in publications. In this publication, our PDGF-C antibody was used as a marker for Non-small Cell Lung Cancer Tumors.

"In univariate analyses, high tumor cell expression of PDGF-B (p
0.001), PDGF-C (p
0.01), and PDGFR-
(p
0.026) were negative prognostic indicators for disease-specific survival."

Donnem, Tom MD, Al-Saad, Samer MD, Al-Shibli, Khalid MD, Andersen, Sigve MD, Busund, Lill-Tove MD, PhD, Bremnes, Roy M. MD, PhD. Prognostic Impact of Platelet-Derived Growth Factors in Non-small Cell Lung Cancer Tumor and Stromal Cells. Journal of Thoracic Oncology. 3(9):963-970, September 2008.

Featured Reagent:
PDGF-C

Related Reagents to Consider:
PDGF R Alpha
PDGF R Beta
VEGF/Flt-1
EGF
Cancer Antibodies
All Neurotrophin and Growth Factor Antibodies
Platelet Derived Growth Factor Proteins
Cancer Research Proteins
Neurotrophin and Growth Factor Proteins

Rat Sensory Neurons and NK-1

A publication featuring our Neurokinin-1 (NK 1) Receptor just crossed my radar. It is authored by our friend, Dr. Matt Ramer.

Matt and his team at University of British Columbia study primary sensory nerve cells (neurons), which are responsible for the transmission of somatic (bodily) sensations such as touch, pain, hot, cold and so on from the periphery (skin, muscles and viscera) to the central nervous system (CNS, spinal cord and brain). His research extends to therapeutic potential of neurotrophins on regeneration in spinal cord injury and deafferentation pain.

In the past several years, he has generouly shared NT-3 and BDNF IHC data with us.

Here's a link to the publication:

Matt Ramer. Anatomical and functional characterization of neuropil in the gracile fasciculus. The Journal of Comparative Neurology. 10.1002/cne.21785.

Featured Reagent
Neurokinin-1 (NK 1) Receptor

Other Reagents to Consider:
Neurokinin-1 (NK 1) Human (RA25003)
Neurokinin-3 (NK 3) (RA25002)
Substance P (GP14103)
proNeurokinin B (RA25008)
All Neuropeptides
All Pain and Inflammation

ELISA techniques and protocols

Power Point of ELISA Methods and Protocols(pps - 1.4M)
This teaching guide covers the three major types of ELISA: indirect, competitive, and sandwich. It integrates theory with practice, to help you understand what you are doing, and help you to do it!

Courtesy of our friends at Novus Biologicals.

Ischemia, Inflammatory Response and Umbilical Stem Cells

We would like to send Kudos to Dr. Yan Xu and his colleagues at University of Pittburgh for their findings on inflammatory response in Golbal Ischemia. Their work was recently published:

Aaron Hirko, Renee Dallasen, Sachiko Jomura, Yan Xu. Modulation of Inflammatory Responses after Global Ischemia by Transplanted Umbilical-Cord Matrix Stem Cells. Stem Cells First published online August 21, 2008; doi:doi:10.1634/stemcells.2008-0075

Secondary to Cardiac Arrest is Brain Damage do to lack of blood flow. This is marked by a delayed loss of Neurons in CA1 hippocampus region of the brain due to inflammatory response.

The story timeline of this response is good then bad with interesting twists. The delay in neuronal loss is linked to initial inflammation. It involves both reactive astrocytes (astrocytosis) and glia. Delaying the loss is, of course, good.

...But then, the reactive astrocytosis and related glial scarring cause a physical and biochemical barrier to regeneration of neurons...a bad thing. Protecting the microglia is a good thing, because they these cells serve as scavengers for clearing the cellular debris. They can also secrete a variety of cytotoxic and protective chemicals.

The wow factor in this research is that implanted rat umbilical-cord matrix (RUCM) cells can provide partial protection against neuronal injury in rat brains. Rats treated with RUCM cells three days prior to an 8-min CA had only 25-32% neuronal loss in the hippocampal CA1 region compared to the typical 50-68% neuronal loss observed in the untreated or the vehicle-treated animals. This could be due to to the favaorable modulation of the "good-bad" inflammatory response.

The good news in the search for therapies for stroke and cardiac arrest victims is combined, stem-cell-like RUCM cells offer protection against neuronal injury after global cerebral ischemia by enhancing the survivability of the astroglia in the selectively vulnerable regions.

We are pleased that the research team used our GFAP antibody as an marker for astrotytic in their studies.

Neurotoxicity Testing

We combine our expertise in providing fresh and healthy:

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

AND Apoptosis Research Reagents

to help researchers more effectively study Neurotoxicity.

Images: Polycaspase Assay Kit, green was used to assess cell death in primay rat hippocampal neurons.Cells were plated on 25-mm poly-l-lysine-coated coverslips at 300,000 cells per coverslip. Cells were used at 4 or 8 days in vitro. Composite imagae (A) 3 out of 4 cells are apoptotic (green). No cells were necrotic as both of the PI-positive cells were FLICA-positive; they had compromised membranes and were probably in the late stages of apoptosis rather than necrosis. (B) 3 Caspase-positive cells fluoresce green.

No Pain; No Gain

We work hard to make sure our Pain and Inflammation antibodies continue to be a gold standard for researchers. We follow up with virtually all researchers to make sure they work to expecations in each unique application.

We also look for references in current publication. Although published in 2006, this one just crossed our radar scope.

It contains multiple images of 3 of our top sellers: Mu Opioid Receptor, VR1 C-terminus (TRPV1) and VR1 N-Terminus (TRPV1).

Shao-Rui Chen and Hui-Lin Pan. Loss of TRPV1-expressing Sensory Neurons Reduces Spinal : Opioid Receptors but Paradoxically Potentiates Opioid Analgesia. J Neurophysiol (February 8, 2006). doi:10.1152/jn.01343.2005

Transthyretin, αAPP peptides and Alzheimer's Disease

Demographics point to increasing rates of Alhzheimer's Disease. This disease steals away the golden years of sufferers. It also costs society billions healthcare.

Research for the cure marches on. Researchers know the disease is characterizedby the deposition of amyloid β-peptide (A-Beta) in the brain. The challenge is finding a way to protect the brain from these depositions or reverse the process.

Dr. Isabel Cardoso and her team at Instituto de Biologia Molecular e Celular have recently published compelling work in this area. Here they provide more important evidence for the role of a Transthyretin (TTR) protective mechanism. This mechanism could include the removal of deposited A-Beta.

Rita Costa, Frederico Ferreira-da-Silva, Maria J. Saraiva, and Isabel Cardos. Transthyretin Protects against A-Beta Peptide Toxicity by Proteolytic Cleavage of the Peptide: A Mechanism Sensitive to the Kunitz Protease Inhibitor.
PLoS ONE. 2008; 3(8): e2899. Published online 2008 August 6. doi: 10.1371/journal.pone.0002899.

A-Beta proteolysis by TTR is KPI-sensitive.
A- A-Beta incubated with TTR (A-Beta+TTR) shows a weaker A-Beta monomer band as compared to A-Beta alone (A-Beta), indicative of proteolysis, as analyzed by SDS-PAGE electrophoresis followed by western blot. Pre-incubation of TTR with pefabloc (A-Beta+(TTR+pefabloc)) and with an αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI+−APP)) inhibits TTR proteolytic activity, whereas the αAPP peptide without the KPI domain (A-Beta+(TTR+KPI−−APP)) facilitates proteolysis. B- % of inhibition of TTR proteolysis by quantification of band intensity in A. C- Ultrastructural analysis by TEM of preparations incubated for 15 hours, as described in Materials and Methods. TTR inhibited A-Beta aggregation as compared with A-Beta incubated alone (upper panels). Pre-incubation of TTR with αAPP peptide containing the KPI domain (A-Beta+(TTR+KPI+−APP)) abrogated TTR ability to avoid A-Beta aggregation, whereas αAPP lacking the KPI domain (A-Beta+(TTR+KPI−−APP)) did not affected TTR activity (lower panels). Scale bar=500 nm.

Neuromics' Reagent Used-APP 228

MMP-9, E. Coli and Breast Cancer

The application of live bacteria for cancer therapies holds promise. In this study, Escherichia coli K-12 colonization on the tumour microenvironment by immunohistochemistry and fluorescence microscopy in the murine 4T1 breast carcinoma model. MMP-9 and TNF-alpha expression is altered in the sites of tumors.

Interestingly, the authors observed a postive change in the expression of these proteins after colonization.

Stephanie Weibel, Jochen Stritzker, Matthias Eck, Werner Goebel, and Aladar A. Szalay. Colonization of experimental murine breast tumours by Escherichia coli K-12 significantly alters the tumour microenvironment. 10.1111/j.1462-5822.2008.01122.x. Cellular Microbiology. © 2008 Blackwell Publishing Ltd
...goat anti-mouse MMP-9 antibody (Neuromics, Edina, MN)...

Parkinson's Disease Research Blog: Mitochondria or not, a reply

Parkinson's Disease Research Blog: Mitochondria or not, a reply

Cholesterol Homeostasis and Diabetes

Hormone-sensitive lipase (HSL) is widely expressed in adipose tissue. Interestingly, HSL-null mice have been shown to be resistant to diet-induced obesity. Despite this characteristic, they can also show insulin resistance. This resistance is contributing factor in type 2 diabetes.

Scientists have also shown that pancreatic beta cells, responsible for insulin release, begin to malfunction when their cholesterol levels build up. So what is the link?

The study referenced here suggests that HSL plays a critical role in the hydrolysis of cytosolic cholesteryl esters and that increased levels of hepatic cholesteryl esters, due to lack of action of HSL in the liver, is the main mechanism underlying the imbalance in cholesterol metabolism in HSL-null mice.

Celine Fernandez, Marie Lindholm, Morten Krogh, Stéphanie Lucas, Sara Larsson, Peter Osmark, Karin Berger, Jan Boren, Barbara A Fielding, Keith N. Frayn and Cecilia Holm. Disturbed cholesterol homeostasis in hormone-sensitive lipase null mice. Am J Physiol Endocrinol Metab (July 29, 2008). doi:10.1152/ajpendo.90206.2008.

...Liver samples were homogenized in lysis buffer pH 7.0 containing 25 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton and 1X protease inhibitor cocktail (Complete Mini, Roche). Total protein concentration was measured by BCA assay (Pierce) and 50 μg of protein were subjected to SDS-polyacrylamide gel electrophoresis (8 % polyacrylamide). After transfer to PVDF membranes (Invitrogen), blots were incubated with a primary antibody mouse anti-mouse/rat ABCA1 (Neuromics) according to the instructionsof the manufacturer. As secondary antibody a horseradish peroxidase-conjugated sheep antimouse IgG was used. Western blot analysis was performed using a chemiluminescence system (Luminol) and detection was made using a CCD-camera (LAS 1000, Fuji). Band intensities were quantified using the ImageJ software (http://rsb.info.nih.gov/ij)...

MMP-9 and Neuroinflammation & Autoimmunity

Zhi-Yuan Zhang, Zhiren Zhang, Uwe Fauser and Hermann J. Schluesener. Improved outcome of EAN, an animal model of GBS, through amelioration of peripheral and central inflammation by minocycline. DOI. 10.1111/j.1582-4934.2008.00333. © 2008 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.
... Metalloproteinase 9 (MMP-9); 1:500; Neuromics, Edina, MN, USA...

Related Reagents:

proMMP-7 (Human)
MMP-24 (MT5-MMP, Human)
Cancer Antibodies
Neurodegenerative Disease Antibodies
Pain and Inflammation

New Neurotensin Receptor (NTS-1) antibody

Our customers have had success with these antibodies.

Here's a representative publication:

Geneviève Roussy, Marc-André Dansereau, Louis Doré-Savard, Karine Belleville, Nicolas Beaudet, Elliott Richelson and Philippe Sarret. Spinal NTS1 receptors regulate nociceptive signaling in a rat formalin tonic pain model. doi:10.1111/j.1471-4159.2007.05205.x

We are pleased to announce the release of a chicken Neurotensin Receptor 1 (NTS1).

Image: Immuofluorescent detection of NTS1 in cell bodies of neurons in rat dorsal root ganglia fluorescence).

TRPV1 Staining of Mouse DRG

This excellent staining comes to us courtesy of Katharina Zimmermann (Childrens Hospital Boston, Clapham Lab). This is some of the best staining we've seen using our VR1 C-Terminus (TRPV1) - mouse specific antibody.

Images: TRPV1 staining of C57BL/6 mouse dorrsal root ganglia. Tissues were stained using Alexa Fluor© 488 (Green) and counterstained with DAPI (blue).

cryosections, 10 microns thickness

Stressed Induced Analgesia (SIA) and OFQ

This is to highlight excellent work done by our friends at SRI and AfaSci.

Our Orphanin FQ ab was used in experiments by the team. Here's the latest publication highlighting their work:

Xinmin Xie, Jonathan P. Wisor, Junko Hara, Tara L. Crowder1, Robin LeWinter, Taline V. Khroyan, Akihiro Yamanaka, Sabrina Diano, Tamas L. Horvath, Takeshi Sakurai, Lawrence Toll and Thomas S. Kilduff. Hypocretin/orexin and nociceptin/orphanin FQ coordinately regulate analgesia in a mouse model of stress-induced analgesia. J. Clin. Invest. 118(7): 2471-2481 (2008). doi:10.1172/JCI35115. Copyright © 2008, The American Society for Clinical Investigation.

...Brain sections (10 μm) were treated with 0.3% H2O2 to quench endogenous peroxidases and then incubated overnight in primary anti-N/OFQ (1:5,000; RA10106, anti-FGGFTGARKSARKLANQ, Neuromics) and anti-orexin-B (1:5,000; sc-8071, Santa Cruz Biotechnology Inc.) antisera at 4°C with agitation. Sections were incubated in blocking buffer for 1 hour, followed by a 2-hour incubation at room temperature in secondary antisera (Alexa Fluor 546 donkey anti-goat [1:750] and 488 donkey anti-rabbit [1:500]; Molecular Probes, Invitrogen). As a negative control, additional sections were treated similarly, but the primary antibody was omitted. Preadsorption with the N/OFQ peptide FGGFTGARKSARKLANQ was used as a positive control and blocked all specific staining as also found by others...

N/OFQ-containing fibers innervate Hcrt neurons, and N/OFQ inhibits Hcrt neuronal activity. (A) Left panel shows confocal image of N/OFQ-immunoreactive fibers in the vicinity of, and in putative contact with, Hcrt-immunoreactive neurons in the PLH of WT mice. N/OFQ (green) fibers are in close proximity to Hcrt-immunoreactive (red) cells. The arrow indicates the N/OFQ innervation of an Hcrt cell body. Middle panel shows light micrograph of a light brown immunolabeled Hcrt neuron contacted by a dark black bouton (arrow) representing immunolabeling for N/OFQ. Right panel shows electron micrograph taken from ultrathin sections of the same labeled terminal and dendrite shown in the light micrograph in the middle panel. Black arrow indicates the N/OFQ-immunolabeled axon terminal in synaptic contact (red arrowhead) with the dendrite of the Hcrt cell. Scale bars: 10 μm (left and middle panels); 1 μm (right panel). (B) Under current clamp, bath application of N/OFQ (1 μM) hyperpolarized Hcrt neurons, decreased input resistance, and blocked spontaneous firing of action potentials. The resting potential of this cell was –54 mV and was manually adjusted to –60 mV with DC current. Membrane resistance was monitored using hyperpolarizing current pulses (–0.3 nA, 800 ms) delivered every 5 seconds throughout the experiment. (C) Under voltage-clamp mode at a Vh of –60 mV, N/OFQ (1 μM) induced an outward current (–53 pA) in an Hcrt neuron. Notice that the frequency but not the amplitude of the miniature synaptic currents (inward currents) recorded in the presence of TTX (0.5 μM) was also reduced.

TH-Marker for Diabetic Neuropathy

New Pub Referencing our Tyrosine Hydroxylase.

Blount, Andrew L. B.A.; Peled, Ziv M. M.D.; Dexter, Erica L. B.S.; Nagle, Raymond B. M.D., Ph.D.; Maloney, Christopher T. M.D.; Dellon, A Lee M.D., Ph.D. Sympathetic Nerves in the Tarsal Tunnel: Implications for Blood Flow in the Diabetic Foot. Plastic & Reconstructive Surgery. 122(1):188-191, July 2008.

...Specimens were fixed in 10% neutral buffered formalin for 3 to 4 hours and then embedded in paraffin. Three-micron sections were obtained from the block and used for immunohistochemistry, which was performed using a mouse monoclonal antibody against tyrosine hydroxylase (MO20001; Neuromics, Edina, Minn.) diluted 1:50 in Discovery Diluent (Ventana Medical Systems, Inc., Tucson, Ariz.) on a Discovery XT Automated Immunostainer (Ventana Medical Systems)...
Images: (A) Tyrosine hydroxylase immunohistochemical staining of tibial epineurium from the tarsal tunnel. Positively staining sympathetic fibers (arrows) are seen within a larger nerve bundle. (B) Tyrosine hydroxylase immunohistochemical staining of connective tissue from the tarsal tunnel. Several positively staining sympathetic fibers (arrows) are seen innervating the media of a local venule.

SP is Back and Better than Ever

We have successfully re-made our widely used Guinea Pig Substance P antibody. It has been tested both internally and by an interested customer. The results exceeded expectations.

This antibody has proven useful for Pain/Inflammation and Diabete/Obesity Research.

Image: Immuofluorescent detection of Substance P in rat spinal cord dorsal horn (red fluorescence). DAPI (blue) was used as counter stain.

Here're are related publications:

P . Tsai , A . Alonso , M . Pinto , D . Leigh , E . Weiler , J . Fricton , M . Erickson , L . Stone , L . Kehl. Substance P is co-localized with protein gene product 9.5-immunoreactive nerve fibers in intervertebral discs from patients with painful degenerative disc disease. doi:10.1016/j.jpain.2006.01.096
...guinea pig anti-substance P (1:500; Neuromics, Edina, MN) ...
Mei Bigliardi-Qi, Claire Gaveriaux-Ruff, Katrin Pfaltz, Pierre Bady, Tommy Bauman, Theo Rufli, Brigitte L Kieffer and Paul L Bigliardi. Deletion of - and -Opioid Receptors in Mice Changes Epidermal Hypertrophy, Density of Peripheral Nerve Endings, and Itch Behavior. Journal of Investigative Dermatology (2007) 127, 1479–1488. doi:10.1038/sj.jid.5700661; published online 21 December 2006.
.. substance P (Neuromics 1:200) staining, the sections were incubated at 4°C over night in Zamboni buffer. ...

Related Reagents:
Neurokinin-1 (NK 1) Receptor
Neurokinin-1 (NK 1) Human Receptor
Neurokinin-3 (NK 3) Receptor
proNeurokinin B (proNKB or P2)
All Neuropeptides
Pain and Inflammation Antibodies
Diabetes and Obesity Antibodies
Substance P Customer Data

ELISPOT

We will be releasing soon a website dedicated to ELISA and ELISpot.

Here are excellent "how to" videos.

Molecular Pathways and Heart Development

Owen WJ Prall, Mary K Menon, Mark J Solloway, Yusuke Watanabe, Stéphane Zaffran, Fanny Bajolle, Christine Biben, Jim J McBride, Bronwyn R Robertson, Hervé Chaulet, Fiona A Stennard, Natalie Wise, Daniel Schaft, Orit Wolstein, Milena B Furtado, Hidetaka Shiratori,6 Kenneth R Chien, Hiroshi Hamada,6 Brian L Black, Yumiko Saga, Elizabeth J Robertson, Margaret E Buckingham, and Richard P Harvey. An Nkx2-5/Bmp2/Smad1 negative feedback loop controls second heart field progenitor specification and proliferation. Cell. 2007 March 9; 128(5): 947–959. doi: 10.1016/j.cell.2007.01.042.

Summary: During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were up-regulated, leading initially to progenitor over-specification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.

...goat anti-Isl1, raised against full length human Isl1, GT15051, Neuromics)...

Combined Neuron Cultures Assays

We would like to thank Dr. Matteo Porotto, Weill Cornell Medical College

Larger Image

Primary rat neurons: 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

Primary Neuron Cultures Featured
Combined Hippocampus, Cortex, and Ventricular -E18
Other Reagents to Consider
All Primary Neurons/Astrocytes, Stem Cells and Media
Transfection Reagents
Neuron-Glial Markers
Stem Cell Markers