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Saturday, April 19, 2014

Extracellular Matrix Environment and Chemotherapeutics

Substrate Matters!

Neuromics' has been promoting a variety of 3-D Cell Based Assay Solutions for the past several years. These include: Nanofibers, Hydrogels and Extracellular Matrix (ECM) Proteins. We have found that the adoption rate for these as standard tools for drug discovery is slower that we anticipated.

We believe substrate matters so I am pleased to share a recent publication that references use of our Collagen IV and other ECM proteins. This confirms the importance of using a more in vivo like environment in testing chemotherapeutics: Thuy V. Nguyen,Marianne Sleiman,Timothy Moriarty,William G. Herrick,Shelly R. Peyton. Sorafenib resistance and JNK signaling in carcinoma during extracellular matrix stiffening. Publication: Biomaterials. Elsevier. 13 April 2014. http://dx.doi.org/10.1016/j.biomaterials.2014.03.058.

Abstract: Tumor progression is coincident with mechanochemical changes in the extracellular matrix (ECM). We hypothesized that tumor stroma stiffening, alongside a shift in the ECM composition from a basement membrane-like microenvironment toward a dense network of collagen-rich fibers during tumorigenesis, confers resistance to otherwise powerful chemotherapeutics. To test this hypothesis, we created a high-throughput drug screening platform based on our poly(ethylene glycol)-phosphorylcholine (PEG-PC) hydrogel system, and customized it to capture the stiffness and integrin-binding profile of in vivo tumors. We report that the efficacy of a Raf kinase inhibitor, sorafenib, is reduced on stiff, collagen-rich microenvironments, independent of ROCK activity. Instead, sustained activation of JNK mediated this resistance, and combining a JNK inhibitor with sorafenib eliminated stiffness-mediated resistance in triple negative breast cancer cells. Surprisingly, neither ERK nor p38 appears to mediate sorafenib resistance, and instead, either ERK or p38 inhibition rescued sorafenib resistance during JNK inhibition, suggesting negative crosstalk between these signaling pathways on stiff, collagen-rich environments. Overall, we discovered that β1 integrin and its downstream effector JNK mediate sorafenib resistance during tumor stiffening. These results also highlight the need for more advanced cell culture platforms, such as our high-throughput PEG-PC system, with which to screen chemotherapeutics.



Figure: High-throughput biomaterial platform for drug screening. (A) The high-throughput platform consists of a black-walled, glass bottom plate, with PEG-PC gels cast in each of the inner 6x10 wells. (B) Gels can be functionalized with any protein or peptide of interest, and they support the adhesion and growth of carcinoma cells. We used this platform to test carcinoma cell response to a kinase inhibitor (sorafenib) as a function of underlying gel stiffness and ECM adhesive protein cocktail. (C) A representative graph of SkBr3 proliferation (y-axis) in response to sorafenib (x-axis) across a range of gel stiffness (colors) demonstrates the IC-50 calculation.

This confirms the importance of considering your substrate environment when developing your in vitro assays for High Content and High Throughput Drug Discovery. We will continue to provide updates.

Thursday, April 17, 2014

HIV-1R Viral Protein R and Memory Impairment

Using Synpatophysin as Marker for Synaptic Loss

Our Neuron-Glial Markers continue to shine in challenging applications. Here researchers examined whether infusion of the Vpr-expressing astrocytes affected synaptophysin expression in the hippocampus. The authors of the study, using Neuromics' Mouse Monoclonal Synaptophysin Antibody,  found a significant reduction in synaptophysin staining in CA3: Lilith Torres and Richard J Noel. Astrocytic expression of HIV-1 viral protein R in the hippocampus causes chromatolysis, synaptic loss and memory impairment. Journal of Neuroinflammation 2014, 11:53 doi:10.1186/1742-2094-11-53.

Images: Astrocytic HIV-1 viral protein R (Vpr) expression decreased synaptophysin immunoreactivity (A) Representative light photomicrograph showing the distribution of synaptophysin immunoreactivity in the rat hippocampal CA3 formation. Green fluorescent protein (GFP) right side. Vpr shows both left and right. Magnification 100×. (B) Densitometric analysis revealed significantly decreased mean value for the Vpr group compare to control.


Protocol: To examine changes in synaptophysin between control and HIV-1 Vpr exposed rats, tissue sections from each group were processed for immunocytochemistry. The samples were cut at 4 μm thickness with a microtome (Microm HM340, Microm International) and fixed to positively charged microscope slides. Fixed tissues were deparaffinized in xylene substitute for 30 minutes, rehydrated through graded alcohols and neutralized with 3% hydrogen peroxide (Sigma-Aldrich), followed by a rinse under running tap water and immersion in antigenretrieval solution (0.01 M citrate, pH 6.0) for 1 minute at 98°C. Then sections were washed in TBS for 5 minutes and treated with blocking solution containing normal goat serum (BioGenex, cat# HK112-9KE). Sections were incubated for 24 hrs at 4°C in mouse monoclonal antisynaptophysin antibody (Neuromics, cat # MO20000, 1:500 dilutions). Negative controls with TBS instead of primary antibody were run in each slide. Primary antibody was washed in TBS buffer for 2 × 5 minutes and incubated with Multi Link secondary antibody (Super Sensitive Link-Label IHC Detection System, cat# LP000- ULE, BioGenex, San Ramon, CA, USA). Secondary antibody was washed in TBS and incubated in ABC-HRP, washed in TBS buffer and incubated in 3,3′-diaminobenzidine (cat# HK153-5KE, Biogenex, San Ramon, CA, USA). Slides were rinsed in water and counterstained with hematoxylin for 30 sec. The sections were rinsed, dehydrated and and mounted with Cytoseal XYL (cat# 8312-4, Richard Allan Scientific, Kalamazoo, MI, USA). For quantitative densitometry, images of regions of interest (ROI) in the CA3 were captured from 5 rats in each group using NIH Image J 1.50 software.

We will continue to work hard to fill all your Neuroscience Research Needs.

Friday, April 04, 2014

Osteoblast Activators and Musculoskeletal Disease Assays

We now have the ability to precisely test human Osteoblast activating agents. Tools available include:

  1.  Live cell screening
  2. Quantibody Bone Metabolism Arrays
  3. PCR
  4. Flow Cytometry
  5. Immunostaining
  6. Western Blotting
This means we can generate data that provides you a clear picture of the effects of small molecules/compounds on the activation, expansion and migration of Osteoblasts. Here's an example.

Image: Activin A expression from Osteoblasts treated with Activators vs Controls

We will soon be releasing a turn-key assay for Osteoporosis Drug Discovery. We can also run custom assays that fit your specific requirements. To learn more, you can call (612-801-1007) or e-mail: pshuster@neuromics.compshuster@neuromics.com). Thank you. Pete Shuster, CEO and Owner, Neuromics.

Wednesday, March 26, 2014

hN2 Primary Human Neurons and Toxicity Assays

Neuromics' hN2 Cells Continue to Shine

I previously posted our Human hN2 Neurons being used for studying the mechanisms of  Nerve Agent VX: hN2 Human Neurons for Toxicity Screening. The versatility of these neurons enabled the researchers to perform a microarray study in which cultured human neural cells were exposed to 0.1 or 10 μM of VX for 1 h. Global gene expression changes were analyzed 6, 24, and 72 h post exposure. Solid primary cell based assay results start with healthy and well behaving cells.

I am pleased to announce success in exposing the cells to a specific cytotoxicity inducing compound (small molecule): Mark RichardsChee Wee PhoonGwendoline Tze Wei GohEng Khuan SengXu Ming GuoCherine Mei Fong TanWoon-Khiong ChanJoel Mun Kin Lee. A New Class of Pluripotent Stem Cell Cytotoxic Small Molecules. Research Article | published 19 Mar 2014 | PLOS ONE 10.1371/journal.pone.0085039.

Image: Cytotoxic agent used in dose-response curve

Figure: Dose response curves for 3 specialized somatic cell lines (MRC-5, human primary neurons and human neonatal cardiomyocytes) treated with JC011.

This curve illustrates the sensitivity of human neurons to toxic agents. 

We plan on continuing to use these and our hNP1 Human Neural Progenitors in kinetic, "in vivo like" assays. These assays will give quantitative data on both growth and differentiation inducing agents as well as specifics on how the cells behave when exposed to toxic agents. I will be posting results here.

This data should be of interested to neuro-disease/disorders basic and drug discovery researchers. We plan on making the assays available to researchers. We currently also do small molecule testing and gene expression analysis studies a CRO offering. To learn more, I can be reached at pshuster@neuromics.com or 612-801-1007.

Sunday, March 23, 2014

Autism, Inflammation and Stem Cell Enhancers

Proving the Therapeutic Value

We have been running Quantibody® Antibody Arrays on blood serum of children diagnosed with Autism Spectrum Disorder (ASD). All reside in areas of heavy industry in Central Europe. All have elevated levels of one or several heavy metals.

These assays are being run as part of our strategy of treating these children with natural stem cell enhancing supplements. Here are the average serum levels of 2 cytokines (IL-6 and TNF-alpha) and 1 related chemokine (CCL3). All of the children had elevated levels vs healthy controls:

Figure: Serum ASD levels vs Healthy Controls (pg/ml)

TNF-alpha and IL-6 promotes the immune/inflammatory response. These two cytokines are guided to sites (including the CNS) of infection or tissue damage by the chemokine CCL-3 and others. In the normal process, the site(s) of immune response are cleaned (the response) of infection and/or damaged tissue and then repaired. The key with autoimmune diseases and disorders, is that that this process becomes a continuous loop; hence, these cytokines and chemokines are elevated. If the loop is broken or down modulated, the the levels of these should decrease.

Mesenchymal stem cells (MSCs) are immunomodulating and anti-inflammatory. We plan on testing candidate substances (all our currently available as natural supplements) on kinetic assays using our umbilical cord blood human mesenchymal stem cells. These will enable us to quantify  cell growth and expansion. We also plan on testing the best candidates on our human neurons to see the effects on cell behavior.

We will then determine safe dosing working with experts in the U.S. and Europe. During treatment, we will again be testing serum to see if the levels of these and other key Cytokines, Chemokines and Growth Factors. This will give proof as to to whether or not the treatments are working. If so, we should see the serum levels of these move toward to those of healthy controls.

I plan on making more data as it becomes available.

Thursday, March 20, 2014

Irritable Bowel Syndrome (IBS) and BDNF

Gene Expression Analysis Determines BDNF's Role in IBS

In this study, Researchers used Neuromics'  i-FectTM Transfection Kit to deliver BDNF to determine effect on visceral hypersensitivity (VHS): J. H. Winston1 Q. Li1, S. K. Sarna1. Chronic prenatal stress epigenetically modifies spinal cord BDNF expression to induce sex-specific visceral hypersensitivity in offspring. Article first published online: 4 MAR 2014. Neurogastroenterology & Motility. DOI: 10.1111/nmo.12326. The Journal of Pain, Volume 14, Issue 11, November 2013, Pages 1485–1491 http://dx.doi.org/10.1016/j.jpain.2013.07.007

Intrathecal treatment with brain-derived neurotrophic factor (BDNF) antagonists reduced VMR to colorectal distension (CRD) in female chronic prenatal stress (CPS)+HeICS rats. (A) Graph shows that intrathecal administration of BDNF antagonist trkBFc in female CPS rats significantly decreased VMR to CRD, 24 h following adult 29 HeICS (two-way repeated measures ANOVA found a significant main effect of treatment, F1,53 = 10.4, p = 0.015; post hoc tests found significant differences at 30, 40, 50, and 60 mmHg, n = 4). (B) Graph shows that intrathecal administration of BDNF siRNA in female CPS rats significantly decreased VMR to CRD, 24 h following adult 29 HeICS (two-way repeated measures ANOVA: treatment 9 pressure interaction, F1,77 = 3.49, p = 0.008, tukey post hoc tests found significance at 30 mmHg, p = 0.013 and at 40, 50 50, 80 mmHg, p < 0.001, n = 7 Ctr., n = 6 BDNF siRNA). (C) Western blot shows a significant decrease in spinal cord BDNF protein expression in rats treated with BDNF siRNA (*p < 0.05).

Conclusion: Chronic prenatal stress followed by a second exposure to HeICS in adult offspring exacerbated visceral hypersensitivity (VHS) greater in female offspring that persisted longer than in male offspring. Chronic prenatal stress up-regulated BDNF expression in the lumbar-sacral dorsal horn that correlated with the exacerbation of VHS in female, but not in male offspring by increasing RNA Pol II binding and histone H3 acetylation, and decreasing histone deacetylase 1 association with the core promoter of BDNF in female offspring.

Thursday, February 27, 2014

Autism, Heavy Metals, BDNF and HSP-70

Autism Spectrum Disorder (ASD) Testing using Custom Quantibody® Array

We recently completed testing blood serum from ASD children. These children were all from Central Europe and lived in cities with heavy industries. All had elevated levels of heavy metals like Aluminum and Copper. We believe that these levels may be contributing to the pathogenesis of the ASD symptoms.

Though the sample was small, the results were striking. The elevated levels of Heat Shock Protein 70 (HSP 70) is consistent with high heavy metal toxicity. Studies have reported both elevated and decreased levels of Brian Derived Neurotrophic Factor (BDNF) vs healthy controls, but is always reduced in subjects with high heavy metal blood serum levels. Could these elevated heavy metals be a root factor ASD Children?


Based on these findings, we have proposed a 2 pronged treatment strategy:
  1. Bring heavy metal serum levels into acceptable balance.
  2. Treat ASD children with natural stem cell enhancing substances
Stem Cells are immune/inflammation supressive and capable of repairing tissue damage. Our theory is that this could result in improvement in ASD related symptoms. Testing serum levels of BDNF, HSP-70 and other select bio-markers would confirm therapeutic efficacy.

I will be posting results updates here so stay tuned.

Tuesday, February 25, 2014

Kinetic Assays and Stem Cell Toxicology Studies

UCB Derived hMSCs and Cobalt

Here're results from a recent Kinetic Assay study we conducted using our Umbilical Cord Blood Derived Stem Cells:

Images and Figure: Images: Dose-Response curve for Co++ toxicity to Hoechst-stained hMSCs (UCB Derived catalog number SC00A1-HC). The bar graph on the lower right shows cell counts verses [Co++] at 24, 48 & 72 hours exposure to Co++. Results at 144 hours showed massive cell death. The initial increase in cell counts at low concentration may reflect the well-known activation of HIFs by Co++. Counts were determined by Hoechst-stained MSCs and simultaneous propidium iodide staining shows increasing numbers of permeable (presumably dead) cells at 24, 48 & 72 hours post-Co++ during exposure to 10% DMSO. Images acquired on Biotek Cytation 3 imaging system.

We are currently designing  assays for testing small molecules and compounds. These are customized for Musculoskeletel Diseases Drug Discovery. They will be released in Q2 2014.

We also offer CRO Services. We have the ability to test different analytes and their impact on: Cell Migration, Differentiation and Expansion. This could include the study of toxic analytes on these behaviors. We are also assaying various supplements that claim to endogenously boost stem cells or other natural substances like Li-VPA. We can do these studies using your stem cells or ours.

Questions or interest? I can be reached at pshuster@neuromics.com or cell: 612-801-1007.

Thursday, February 20, 2014

More MOR Publications

Latest Mu Opioid Publications

Our Mu Opioid Receptor Antibodies have set the standard for the study of Pain Mechanisms. We have posted >40 publications referencing use of these antibodies.

Here's several published in 2014:

Charlie H.T. Kwok, Ian M. Devonshire, Andrew J. Bennett, Gareth J. Hathway. Postnatal maturation of endogenous opioid systems within the periaqueductal grey and spinal dorsal horn of the rat. PAIN - January 2014 (Vol. 155, Issue 1, Pages 168-178, DOI: 10.1016/j.pain.2013.09.0220. ...rabbit anti-MOR (Neuromics, Edina, MN, USA; 1:1000 with tyramide signal amplification protocol)...


Images: Immunohistochemical expression of opioid peptides and receptors in the DH (spinal cord dorsal horn) during postnatal development. (A) POMC (pro-opiomelanocortin) immunoreactivity in the dorsal horn in postnatal day (P)10, P21, and adult rats. White arrows depict where cell staining was found. Interestingly, fibre staining was also observed in the superficial dorsal horn (lamina I) of adult rats, but not in the younger ages. (B) Since both cell and fibre staining were observed, staining intensity was used to quantify the immunoreactivity of POMC in the DH. Quantified staining intensity for POMC in the DH significantly decreased as the animals aged, with highest immunoreactivity found at P10. (C) Enkephalin immunoreactivity in the DH was restricted to the superficial laminae and only fibre staining was observed. (D) Quantified staining intensity for enkephalin illustrate an age-dependent increase in immunoreactivity, which was highest at adult P10.(C) Enkephalin immunoreactivity in the DH was restricted to the superficial laminae and only fibre staining was observed. (D) Quantified staining intensity for enkephalin illustrate an age-dependent increase in immunoreactivity, which was highest at adult. (E) MOR (μ-opioid receptor) immunoreactivity in the DH, cell staining was found throughout the superficial and deeper laminae in all ages. (F) Cell count of MOR staining in the DH, which showed a significant increase as the animals aged (∗∗P<0 .01="" adult="" i="" p21="" vs="">


J. Desroches, J.-F. Bouchard, L. Gendron, P. Beaulieu. Involvement of cannabinoid receptors in peripheral and spinal morphine analgesia ☆ Neuroscience, Volume 261, 7 March 2014, Pages 23–42. http://dx.doi.org/10.1016/j.neuroscience.2013.12.030. ...The floating sections were then incubated in 1% sodium borohydride in PBS for 30 min, rinsed twice with PBS, and incubated for 30 min at room temperature in a blocking solution containing 3% normal goat serum (NGS) and 0.3% Triton X-100 in PBS. The sections were then incubated overnight at 4 °C with the guinea pig anti-MOP primary antibody (cat# GP10106; Neuromics, Minneapolis, MN, USA) diluted 1:1000 in the blocking solution. The floating sections were then washed in PBS and incubated with a goat anti-guinea pig secondary antibody conjugated with Alexa Fluor 488 (Molecular Probes, Invitrogen, Carlsbad, CA, USA) at a concentration of 1:1000 in PBS for 2 h at room temperature...

We will continue posting customer data and publications that give new insights into the mechanisms of pain.

Monday, February 10, 2014

Quantibody Arrays for Tracking Patient Health

Neuromics is working with Dr. Joe Smarda, a renowned Immunologist, to track levels of cytokines in the blood serum of his clients. We have selected RayBiotech's Quantibody® Arrays for these assays. The Clinics in Joe's network treat his clients for autoimmune related disorders.

Our regime is:
  1. Test clients pre-treatment
  2. Treat
  3. Test
  4. Refine treatment
  5. Test 
The specified treatment regime is continued until clients have blood serum cytokine levels that are in the range of our healthy controls. Here's data from our Quantibody® T-helper cell Cytokine Arrays (pre-treatment).

Figures IL-6, IL-1 beta, MCP-1 and PAI1 Array results in 4 clients with Autoimmune related Diseases.

We plan on posting these serial  testing results. They are designed to monitor status and indicate therapeutic effectiveness.

We will also be sharing some of the specific therapies being used. These will include treatments aimed at mobilizing endogenous stem cells. These cells have natural immune suppression/anti-inflammatory properties. Stay tuned.

Tuesday, January 28, 2014

Pot and Pain

The pressure for states to legalize Marijuana for medical and recreational use is building. The tax benefits are self evident.

The debate for many centers of "true medical benefits". That's why research on understanding analgesic pathways is so important. Ironically, this study was conducted by my friends at Université de Montréal and Université de Sherbrooke in Quebec Canada: J. Desroches, J.-F. Bouchard, L. Gendron, P. Beaulieu. Involvement of cannabinoid receptors in peripheral and spinal morphine analgesia ☆ Neuroscience, Volume 261, 7 March 2014, Pages 23–42. http://dx.doi.org/10.1016/j.neuroscience.2013.12.030.

These teams have proven expert is using our Opioid Receptor Antibodies in their pain research. Here's a synopsis:
•Analgesia is the most common feature shared by the cannabinoid and opioid systems.
•The role of the cannabinoid system in the morphine-induced analgesia is uncertain.
•Peripheral and intrathecal morphine analgesia is altered in cnr1KO and cnr2KO mice.
•This attenuation is neither caused by a MOP malfunction nor by its downregulation.


Images: Deletion of the CB1 or CB2 receptors has no effect on the expression of MOP in the spinal cord. Immunofluorescence of spinal MOP revealed that the expression of MOP in laminae I and II of the dorsal horn of the spinal cord did not differ between cnr1WT (A) and cnr1KO (B) mice or between cnr2WT (C) and cnr2KO mice (D).


Observations here further support the existence of interactions between the cannabinoid and opioid systems. The loss of peripheral and spinal morphine analgesia is apparently caused neither by a decrease in MOP spinal expression nor by altered binding properties or G protein coupling of this receptor in cnr1KO and cnr2KO mice. The mechanisms underlying the loss of morphine analgesia are not clear but could include the release of endogenous cannabinoids in structures along the pain pathway or a disrupted endocannabinoid tone.

It is important funding that enables researchers to understand the analgesic pathways of marijuana continues to grow. This research could yield better control of pain with reduced side effects.

Tuesday, January 21, 2014

FGF and Stem Cells-Options Matter

Proven, Potent and Cost Effective Fibroblast Growth Factors (FGF).

Neuromics have a wealth of expertise in Stem Cells, Media and Growth Factors. FGF is an important component of Stem Cell Based Assays. Our goal is to provide an FGF that fits your requirements like "hand in glove".

Here's a small sampling:
ISO-kine bFGF-100% animal free and serum free-is produced in the endosperm tissue of barley grain (Hordeum vulgare), that exhibits up to 50 times less protease activity than E.coli or mammalian cells. Barley seed is void of any human or animal viral contaminants that could jeopardize your cell culture.
Images A: Expression of OCT4 (green) in the ORF group. B: Expression of TRA-1-60 (green) in the ORF group.

...15 million MNCs were seeded per 150 cm2 tissue culture flask. Culture media was alpha MEM supplemented with 10% heat inactivated fetal bovine serum, 1 mM sodium pyruvate, 100 mM HEPES buffer, 1 mM sodium pyruvate, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.29 mg/ml L-glutamine (all from Invitrogen, Mississauga, Ontario, Canada) and 5 ng/ml of basic FGF or FGF-2 (Neuromics, Edina, MN, USA). Plastic adherent MNCs were allowed to attach and proliferate for 7 days before the first media change under normal oxygen tension (21% O2; 95% air) at 37°C in a humidified incubator with 5% CO2...

Images: Histological characteristics of pellets formulated from mono-cultured MCs, mono-cultured BMSCs and co-cultures of MC and BMSCs after a total of 17 days culture in defined serum-free chondrogenic media. (A-B) Safranin O and collagen II immuno-histochemical staining of representative pellets from cells derived from the same donor. Magnification lens × 20; scale bar is 100 μm. Chowdhury et al. BMC Musculoskeletal Disorders 2013 14:216 doi:10.1186/1471-2474-14-216

We are working hard to provide unique and cost effective solutions for your Stem Cell Based Assay Requirements.

Monday, January 13, 2014

TRPV1 and Osteoarthritis Related Pain

Our TRPV Antibodies are widely used and frequently published. Many of these feature TRPVs' role in nociceptive pain. Specifically they play important roles in the detection of noxious stimuli and inflammatory hyperalgesia.

TRPV1 has been implicated in OA pain, both in animal models and by the finding that TRPV1 genetic variants are associated with the risk of symptomatic knee OA in humans: S Kelly, R J Chapman, S Woodhams, D R Sagar, J Turner, J J Burston, C Bullock, K Paton, J Huang, A Wong, D F McWilliams, B N Okine, D A Barrett, G J Hathway, D A Walsh, V Chapman. Increased function of pronociceptive TRPV1 at the level of the joint in a rat model of osteoarthritis pain. Ann Rheum Dis doi:10.1136/annrheumdis-2013-203413.
Methods: Rat spinal cord sections from MIA- and saline-treated rats (n=5/group) (see online supplemental methods) were incubated with a polyclonal guinea pig anti-TRPV1 antibody (1 : 500, Neuromics, Edina, Minnesota, USA catalogue number GP14100) and then with Alexa 568-conjugated goat anti-guinea pig secondary antibody (1:300, Molecular Probes). TRPV1 immunostaining was visualised with a Leica DMRB/DM4000 B fluorescence microscope and images were acquired using Openlab software (PerkinElmer)...




Images: Transient receptor potential vanilloid 1 (TRPV1) immunoreactivity in the spinal cord. TRPV1 immunofluorescence detected in superficial dorsal horn (10× magnification) in rat lumbar (L3–L5) spinal cord at day 28 post-intra-articular injection of saline (A) or mono-iodoacetate (MIA) (B). Minimum and maximum brightness values were altered (32.01 min and 90.14 max) using Image J so as to highlight the area of TRPV1 positive staining. (C) Quantification of TRPV1 immunofluorescence in superficial dorsal horn of spinal cord taken from rats at 14 or 28 days following intra-articular injection of MIA and at day 28 following intra-articular injection of saline. Data are expressed as mean and SEM (n=5 per group).

Clinical trials of oral TRPV1 antagonists have been limited by on-target-induced hyperthermia. Here experimental evidence for increased functional role of TRPV1 at the level of the joint in a model of OA pain and the demonstration that blockade of joint TRPV1 ablates sensory afferent sensitization and pain behaviour support future targeted site-specific investigations of the therapeutic potential of TRPV1 for OA pain associated with synovitis. This could be good news for OA sufferers.

Monday, December 30, 2013

Autism Spectrum Disorder and Biomarkers

We used our IFN-gamma and BDNF Sandwich Elisa Kits to test the levels of these biomarkers in the blood serum of 2 individuals with Austism Spectrum Disorder. We anticipated  that BDNF would be significantly up or down regulated and IFN-gamma up regulated as confirmed by the literature. see 10.1371/journal.pone.0020470 , DOI: 10.1371/journal.pone.0020470 and DOI: 10.1111/acps.12071.

Our testing results showed:
Figure 1: ASD and Biomarker Levels

Based on these initial findings we are developing a custom Quantibody® Antibody Array designed for the fine tuned testing of individuals with ASD. The biomarkers included in the array will be: HSP70, TGF-beta2, 
Caspase-7, IFN-gamma and BDNF.

Figure 2: ASD Markers vs Controls

These panels will enable us to determine the severity of ASD. From the results, we will be working with our collaborators to determine potential cell based therapies for improving the symptom and behaviors of ASD sufferers. As part of the therapeutic regimes the panels can then be used check on progress towards "wellness". 

This a key initiative for us in 2014 so stay tuned.

Tuesday, December 17, 2013

hMSCs and Mesen-X Media in Actions

Customer Feedback on Neuromics' Human Mesenchymal Stem Cells

The demand for these UCB derived hMSCs continues to grow. We do everything we can to insure users' success. This includes replacing cells if there are any issues.

It is always great to get documented feedback. Dr. Rodney Nash, CEO of Javeen Biosciences used their new Mesen-X media to grow the cells. This GMP manufactured media is TOTALLY serum and animal free. It is shipped at room temperature and requires no attachment agents.

Here's Dr. Nash's feedback: We recently used the hMSCs derived from Umbilical Cord Blood. Their performance was nothing less than excellent. We were highly impressed with their morphology and their doubling rate. In addition the cells respond very well to accutase and maintain their performance after passaging. We highly recommend this cell line."

Neuromics' hMSCs grown using Mesen-X Media

The poperties of this Media makes it an excellent solution for drug discovery. Neuromics will be distributing in early 2014 so there will be much more data to follow.

Saturday, December 07, 2013

Biochemistry of Hair Loss

Hair Follicle Stem Cells and the WNT Pathway

This publication hit my Radar because it referenced use of our Goat Polyclonal GFRA1 Antibody.


In this study, Researchers use Tbx18Cre knockin mouse line to ablate the Wnt-responsive transcription factor β-catenin specifically in  at E14.5 during the first wave of guard hair follicle formation. In the absence of β-catenin, canonical Wnt signaling is effectively abolished in cell clusters of precursors for the hair follicle dermal papilla (DP). Sox2+ dermal condensates initiate normally; however by E16.5 guard hair follicle numbers are strongly reduced and by E18.5 most whiskers and guard hair follicles are absent, suggesting that active Wnt signaling in dermal condensates is important for hair follicle formation to proceed after induction. To explore the molecular mechanisms by which Wnt signaling in dermal condensates regulates hair follicle formation, we analyze genome-wide the gene expression changes in embryonic β-catenin null DP precursor cells. We find altered expression of several signaling pathway genes, including Fgfs and Activin: Su-Yi Tsai, Rachel Sennett, Amélie Rezza, Carlos Clavel, Laura Grisanti, Roland Zemla, Sara Najam, Michael Rendl Wnt/β-catenin signaling in dermal condensates is required for hair follicle formation. Developmental Biology, Available online 3 December 2013. http://dx.doi.org/10.1016/j.ydbio.2013.11.023

Could manipulating PDs be the answer for halting hair loss or better yet reverse the process? This research moves us closer.

Sunday, December 01, 2013

In vivo Like Cell Based Assays!

Save 100 USD on our Potent, Pure and Easy to Culture Stem and Progenitor Cells

We continue to design in vivo like behaviors into our  Cell Based Assays.
Our goal is to enable you to make better research decisions earlier
in your discovery process.

These assays are engineered using our proven, potent and easy to culture eSC and Adult Human Stem Cells and Progenitors.

UCB Derived hMSC differentiated to Osteoblast. Note the calcium depositions.

***December Promotions-New Products-Save 100 USD+ 
on Primary and Stem Cells, Media, Markers and Growth Factors***
I'll be posting videos of our in vivo like assays with specific quantitative data here soon.

Sunday, November 24, 2013

Microelectrodes for On-Chip Manipulation of Human Mesenchymal Stem Cells

Amping up Osteogenesis Assays for Drug Discovery

Neuromics and Vitrobiopharma are developing kinetic, differentiation asssays for improving the Drug Discovery process for Osteo-related diseases like Osteoporosis and Osteoarthritis. We have the ability to develop in vivo like assays with quantitative endpoints. The foundation of these assays are potent, easy to culture and cost effective Human Mesenchymal Stem Cells (hMCSCs).

I wanted to share an excellent study using our hMSCs for Osteogenesis Assays: Hsiao, Y.-S., Kuo, C.-W. and Chen, P. (2013), Multifunctional Graphene–PEDOT Microelectrodes for On-Chip Manipulation of Human Mesenchymal Stem Cells. Adv. Funct. Mater., 23: 4649–4656. doi: 10.1002/adfm.201203631. This represents a novel approach for Osteogenesis Assays and illustrates the capabilities of our hMSCs.
Absract: All-solution-processed multifunctional organic bioelectronics composed of reduced graphene oxide (rGO) and dexamethasone 21-phosphate disodium salt (DEX)-loaded poly(3,4-ethylenedioxythiophene) (PEDOT) microelectrode arrays on indium tin oxide glass are reported. They can be used to manipulate the differentiation of human mesenchymal stem cells (hMSCs). In the devices, the rGO material functions as an adhesive coating to promote the adhesion and alignment of hMSC cells and to accelerate their osteogenic differentiation. The poly(L-lysine-graft-ethylene glycol) (PLL-g-PEG)-coated PEDOT electrodes serve as electroactive drug-releasing electrodes. In addition, the corresponding three-zone parallel devices operate as efficient drug-releasing components through spatial-temporal control of the release of the drug DEX from the PEDOT matrix. Such devices can be used for long-term cell culturing and controlled differentiation of hMSCs through electrical stimulation.

Protocols: The hMSCs (Neuromics, Edina, MN) used in experiments were at passage 3-9. Each passage of hMSCs was maintained on the TCPS dishes with a pre-coating of Geltrex reduced growth factor basement membrane matrix (Invitrogen, CIBCO, NY). All hMSCs were maintained in the growth medium, Dulbecco's modified Eagle's medium-low glucose (DMEM-LG) supplemented with mesenchymal cell growth supplement (MSCGM, Lonza) containing L-glutamine, penicillin, and streptomycin, and incubated in an atmosphere containing 5% CO2 at 37 °C. The medium was replenished every 3 to 4 days. For osteogenic differentiation, the hMSCs were cultured in the osteogenesis induction medium, DMEM-LG supplemented with mesenchymal stem cell osteogenesis kit (Chemicon, Cat. No. SCR028), and incubated on the TCPS dishes (control) and test devices. To study the drug release from the devices, hMSCs were cultured in the osteogenesis induction medium in the absence of DEX. The fresh medium was replaced every 2 to 3 days.


Figure: a–d) Osteocalcin expression in hMSCs cultured on rGO–PEDOT microelectrode arrays of various sizes (rGO–PEDOT-20, rGO–PEDOT-50, rGO–PEDOT-100), revealed through immunofluorescence staining. Cells were cultured for 9 days in osteogenesis induction medium in the absence of DEX; drug release was modulated electrically through three cycles of ES (three-day interval); osteocalcin (green) revealed the bone matrix and DAPI (blue) revealed the nucleus. e) Enlarged view of immunofluorescence of osteocalcin expression in hMSCs cultured on rGO–PEDOT-100. Arrows indicated the formation of bone matrix nodules. Scale bar: 100 μm. f) Schematic representation of some bioelectronic features integrated into our rGO–PEDOT devices. doi: 10.1002/adfm.201203631

Conclusion: Authors explored the effect of the dimensions of the rGO–PEDOT microelectrode arrays on the cell spreading and expression of differentiation; whereas the smaller rGO–PEDOT-20 array exhibited better cell alignment ability, the larger rGO–PEDOT-100 exhibited better performance at inducing osteocalcin expression in hMSCs. They also found that using three-zone parallel devices made it possible to release drugs at three points in time. This concept could presumably be extended to release different types of drugs at different lineages on defined patterns. Eventually, such devices might be applicable in tissue engineering and regenerative medicine.

I plan on frequent updates on ours and others development of Osteogenesis Assays for Drug Discovery.

Wednesday, November 20, 2013

Neurotrack Player, Essen Bioscience & Neuromics Neurons

I claim that our solutions are thoroughly tested, validated and research ready. We measure our performance by making sure this claim rings true with our customers.

One way this is confirmed to me is when a partner has success using one of our solutions in a new way. With that I am pleased to update you on Essen Bioscience's application for our primary neurons. Please note the related assays are being demonstrated in their most current webinar.

We will be featuring kinetic and migration assays using our hMSCs, hOsteoblasts and hChondrocytes in the near future. So stay tuned.

Friday, November 01, 2013

SMG-1 and Parkinson's Disease (PD)

Absence of SMG1 protein could lead to PD


A new study has suggested that the absence of a protein called SMG1 - identified as a Regulator of Parkinson's disease-associated alpha-Synuclein-could aid in the development of Parkinson's and other related neurological disorders.

In light of these findings, we believe SMG1 will have increasing importance for PD Researchers. We now have a solid marker for this protein.


Image: Immunoperoxidase of monoclonal antibody to SMG1 on formalin-fixed paraffin-embedded human adrenal gland. [antibody concentration 1.5 ug/ml inset: : Western blot of SMG1 expression in HeLa NE.

Please check out our comprehensive catalog of markers for Parkinson's.

Friday, October 25, 2013

Our Antibody Arrays and ELISA Manufacturing Partner Receives ISO Certification

RayBiotech Awarded ISO 13485: 2003 Certification 

Our partner, RayBiotech, provides us Antibody Arrays and ELISA Kits. This should give potential customers confirmation that this kit are rock solid.

 NORCROSS, GA -- (Marketwired) -- 09/11/13 -- RayBiotech, Inc. announced today that the company has been awarded ISO 13485: 2003 certification with respect to its compliance in the manufacture of in vitro diagnostics kits to be provided to the research community. The ISO 13485 award applies to multivariant antibody arrays, enzyme-linked immunosorbent assays (ELISAs) as well as membrane and glass format arrays and reagents. ISO 13485 is the International Organization for Standardization's certification that the fundamentals of quality management are in place and actively implemented as formal systems within the organization. The ISO 13485 compliance certification is in addition to the company's formal compliance under Good Laboratory Practices (GLP) and Good Manufacturing Practices (GMP), and it further confirms RayBiotech's commitment to quality control and quality assurance for all of its products and services.
Figure: Multiplex antibody arrays-how they work

We will be posting customers feedback on Bazaarify as they becomes available.

Wednesday, October 23, 2013

CIRM Stem Cell Research Updates: Autism syndrome modeled in lab dish points to futu...

CIRM Stem Cell Research Updates: Autism syndrome modeled in lab dish points to futu...: Here's a major stumbling block in developing therapies for human diseases -- ..Here's a major stumbling block in developing therapies for human diseases -- it's hard to find a fix if you don't really know what's wrong. Take autism spectrum disorders. By now doctors are pretty good at identifying signs of the disease, but without access to brain cells researchers don't really know what's going wrong.





This work is being done by Dr. Ricardo Dolmetsch and his team at Stanford. They are particularly interested in understanding how electrical activity and calcium signals control the development of the brain and how this is altered in children with autism spectrum disorders.

I plan on posting more here as part of my featuring the work of key Autism Researchers like Dr. Valerie Hu at GWU. She will also be featured on my "News Behind the Neuroscience News" Blog at www.neuromics.net.

Saturday, October 12, 2013

P2X3 Receptor and Inflammatory Nociception

P2X3 Activates TRPA1, 5-HT3 and 5-HT1A Receptors

Endogenous ATP via activation of P2X3 Receptors contributes to inflammatory nociception in different models, including the formalin injected in subcutaneous tissue of the rat's hind paw. In this study, researchers evaluated whether TRPA1, 5-HT3 and 5-HT1A receptors, whose activation is essential to formalin-induced inflammatory nociception, are involved in the nociception induced by activation of P2X3 receptors on subcutaneous tissue of the rat's hind paw: Suzy Krimon, Dionéia Araldi, Filipe César do Prado, Cláudia Herrera Tambeli, Maria Cláudia G. Oliveira-Fusaro, Carlos Amílcar Parada. P2X3 receptors induced inflammatory nociception modulated by TRPA1, 5-HT3 and 5-HT1A receptors. Pharmacology Biochemistry and Behavior, Available online 8 October 2013. http://dx.doi.org/10.1016/j.pbb.2013.09.017. Our widely used and frequently published P2X3 R Antibody places a central role in measuring the expression of the protein...containing 5% non-fat dry milk at room temperature, followed by incubation with P2X3 rabbit polyclonal antibody (1:500; Neuromics) overnight at 4 °C, rinsed six times with TBST, and then incubated for 40 minutes in goat anti-rabbit IgG peroxidase...
Image: Neuromics' P2X3 R WB Example: Sequence‐specific siRNA‐mediated repression of P2X3. (A) P2X3 mRNA inhibition by 200 nM siRNA duplexes. Twenty‐four hours after transfection of CHO‐rP2X3 cells, P2X3‐specific mRNA was measured with Q‐PCR and plotted as percentage of mRNA detected in the control treated with Oligofectamine alone. Sequences and modifications are shown in Figure 1B and Table 1. (B) P2X3 protein reduction by 200 nM siRNA‐8646/8647, but not by its mismatch analogue siRNA‐MM‐7558/7559 or the unrelated siRNA‐7126/7127. Twenty‐four hours after transfection, protein was extracted and analysed by western blotting. P2X3‐specific immunodetection reveals expression levels as shown below (an average value from two experiments). Time points as indicated at the top. Molecular weights of two glycosylated forms of P2X3 are shown on the left. 

Conclusions: Nociceptive response intensity was measured by observing the rat's behavior and considering the number of times the animal reflexively raised its hind paw (flinches) in 60 min. Local subcutaneous administration of the selective TRPA1, 5-HT3 or 5-HT1A receptor antagonists HC 030031, tropisetron and WAY 100,135, respectively, prevented the nociceptive responses induced by the administration in the same site of the non-selective P2X3 receptor agonist αβmeATP. Administration of the selective P2X3 and P2X2/3 receptor antagonist A-317491 or pretreatment with oligonucleotides antisense against P2X3 receptor prevented the formalin-induced behavioral nociceptive responses during the first and second phases. Also, the co-administration of a subthreshold dose of αβmeATP with a subthreshold dose of formalin induced nociceptive behavior, which was prevented by local administration of tropisetron, HC 030031 or WAY 100, 135. These findings have demonstrated that the activation of P2X3 receptors induces inflammatory nociception modulated by TRPA1, 5-HT3 and 5-HT1A receptors. Also, they suggest that inflammatory nociception is modulated by the release of endogenous ATP and P2X3 receptor activation, which in turn, increases primary afferent nociceptor susceptibility to the action of inflammatory mediators via interaction with TRPA1, 5-HT3 and 5-HT1A receptors in the peripheral tissue.

I will continue to post pain and inflammation related studies that reference the use of our antibodies.

Friday, September 27, 2013

From Zero to 3-D Cell Based Assays in 15 Minutes

Collagel Hydrogels are Designed to Match Your Cell Types.

We are pleased to add Collagel Hydrogels to our 3-D Cell Based Assay Solutions.

The are 3 different gel types that mimic the different in vivo extracellular environment that your cells experience. You will be able to get really nice adipogenesis with MSCs using our CollaGel Hydrogel Standard. CollaGel Hydrogel Soft is an ideal matrix for growing fibroblast; primary hepatocyte cultures and great for growing smooth muscle cells. CollaGel Hydrogel Soft+ is an ideal matrix for growing nervous cells, veins cells and Hydroxyapatite crystals.

Image: Primary hepatocyte culture in 3D model using our CollaGel Hydrogel. 

Using these gels, you can now form small tube-like structures in combination with Human Umbilical Vein Endothelial Cells (HUVEC) in a 3D model. Our CollaGel Hydrogel can also be used for 3D printing without worrying about the needles in your machine been broken due to the fast gelling process. Collagel Hyrogels can be used for: Stem Cell Behavior Studies Fibrosis Studies,  Cosmetic Toxicology, Hepatocyte Assays, Neuronal Branching, Wound Healing Assays, Cell Invasion Assays, Migration Assays, Cancer Cell Phenotyping and  Bioprinting.

I will be posting Collagel Hydrogel related data and images provided by our customers.

Thursday, September 19, 2013

New Astrocyte-Glial Markers

More Options!

We get a lot of request for markers that will more specifically stain astrocytes, glia and microglia. Let's say, for example, you want to pinpoint these cell types in a mixed neuron-glial culture. You can now do a dual label and generate these results.

Image: Neuron-glial cell mixture cultures stained with  ALDH1L1 (red) and our monoclonal antibody against GFAP (green). Blue is a DNA stain. ALDH1L1 stains astrocytes and excludes from neuron cells. ALDH1L1 stains the astrocytes cell body and processes, whereas GFAP labels the intermediate filament of the cytoskeleton in subset of astrocytes. Astrocytes are positive for both ALDH1L1 and GFAP appear yellow. ALDH1L1 also labels many astrocytes not labeled by GFAP, which appear as red. Inset:Blot of rat liver tissure homogenates blotted with ALDH1L1. The antibody binds strongly a band at ~100 kDa.

Or how about these results:
Image: Mixed neuron-glial cultures stained with Mouse Monoclonal GFAP, and Chicken Polyclonal Neurofilament-NF-L (green). The GFAP antibody stains the network of astrocytes in these cultures, while the NF-L antibody stains neurons and their processes. The blue channel shows the localization of DNA. This antibody also works on formalin fixed paraffin embedded brain tissues. Inset: Strip blot of rat spinal cord protein extract stained with GFAP. A prominent band at about 55 kDa corresponds to the major isoform of GFAP.

We will continue to post new additions to our Neuronal-Glial Markers.

Sunday, September 15, 2013

Antibody Array-ELISA Kits for Cancer Researchers

We continue to build our catalog of Antibody Arrays & ELISA Kits
for Cancer Researchers. Our RayBio® C-Series Ctyokine Arrays
enable you to analyze the expression of up to 120 Cytokines for about
the price of a single ELISA (585 USD).

 Antiboy Array 5
Figure 3. PTEN Downregulation in HER2-Overexpressing Cells Activates an IL6/NF-kB-Mediated Inflammatory Feedback Loop (A–E) MCF7-HER2+PTEN cells secreted 3- to 5-fold higher levels of IL6, IL8, and CCL5 compared to MCF7-HER2+ or MCF7-PTEN cells as determined by RayBio human cytokine antibody Array 5 (A). The intensity of each blot compared to control was determined by Kodak image analyzer (B) and confirmed by ELISA (C). Downregulation of PTEN in HER2-amplified breast cancer cells, BT474, SKBR3, HCC1954, and Sum159-HER2+ (D) results in increased levels of these cytokines in vitro (E). http://dx.doi.org/10.1016/j.molcel.2012.06.014

Array Capabilities:
  • Detects expression of 96 secreted proteins in a single sample, in a single day. 
  • If you can do a Western, you can use a C-Series Antibody Array. And get excellent results the first time!  
  • Sandwich ELISA pairs give C-Series arrays high sensitivity and specificity.  
  • C-Series arrays are compatible with practically ANY liquid sample. 
References have cited using membrane-based C-Series Cytokine Antibody Arrays with nearly every liquid sample type imaginable, including: cell-cultured and co cell cultured media, cell and tissue lysates, tissue/organ perfusates, serum, plasma, urine, and many other body fluids, including cyst, interstitial, synovial, blister, cerebrospinal, prostatic and amniotic fluids, as well as abscesses, broncho-alveolar lavage, sputum, saliva, tears, breath condensates, and even human milk and colostrum. Publications referencing use of our Antibody Arrays.

I will be posting more new developments here.

Monday, September 09, 2013

Stem Cell Markers

Our Stem Cell Reagents our widely used and frequently published. We are proud of the positive feedback on our Stem Cell Markers.
Image: Tuj-1 staining of Neuron-specific class III β-tubulin in differentiated human neural progenitor cells. Cells were stained using goat anti-mouse Alexa Fluor 488 (green) secondary antibody (Molecular Probe, A-11001) and counterstained with PI (red).
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).

Tuj-1: Customer Publications and Data.

I will continue to post updates.

Tuesday, August 20, 2013

Proud Distributor of RayBiotech's Antibody Arrays and ELISAs

Pioneer and Market Leader

I am pleased to announce we are a distribution partner for RayBiotech. The solutions we offer include Antibody Arrays and ELISA Kits. With these additions, you now have many options for the either the qualitative, semi-quantitative or quantitative measurement of protein expression. From single antibody pair ELISAs to multiplexed arrays. The arrays enable you to simultaneously analyze up to 80 cytokine, inflammatory response, growth factor or angiogenesis related proteins.

In this posting, I focus on our new C-Series Antibody Array Kits. The kits utilize the sandwich immunoassay principle, wherein a panel of capture antibodies is printed on a nitrocellulose membrane solid support (usualy 2.5 cm x 3 cm). The array membranes are processed similarly to a Western blot (chemiluminescent readout). Signals are then visualized on x-ray film or a digital image, allowing densitometry data collection and calculation of fold-changes for each detected protein. The entire procedure can be completed in 1 day, and is simple enough to permit even the novice researcher to successfully collect data with very few pitfalls and little or no optimization.

They are proven and frequently published. You can now analyze the protein expression in you assays for a fraction of the cost of tradition methods. Here's an example: Simona Giorgini, Daniela Trisciuoglio, Chiara Gabellini, Marianna Desideri, Laura Castellini, Cristina Colarossi, Uwe Zangemeister-Wittke, Gabriella Zupi and Donatella Del Bufalo. Modulation of bcl-xL in Tumor Cells Regulates Angiogenesis through CXCL8 Expression. doi: 10.1158/1541-7786.MCR-07-0088. Mol Cancer Res August 2007 5; 7

Figure: bcl-xL overexpression in ADF glioblastoma and M14 melanoma cells increases CXCL8 expression. Image of membranes from a protein array. Membranes are probed with CM from the (A) glioblastoma control (AN8) and bcl-xL–overexpressing (AXL42 and AXL74) clones and (B) melanoma control (Mneo) and bcl-xL–overexpressing (MXL12 and MXL90) clones. In the negative control (NEG), the CM is replaced with an appropriate mock buffer according to array protocol. The intensity of protein signals for each membrane was compared with the relative positive signals by densitometric analysis. C. Schematic representation of proangiogenic factors that can be detected by the use of the membrane. The CXCL8, TGF-1β, TIMP1, TIMP2, and VEGF protein signals (two spots) are indicated by red, blue, yellow, violet, and green rectangles, respectively, in each image....The Human Angiogenesis Antibody Array I (RayBiotech. Inc.) was used according to the manufacturer's protocol to evaluate the secretion of 20 angiogenic factors into the CM of the different lines. A schematic representation of the proangiogenic factors that may be detected by the use of the array has been reported in the above figure. Membranes spotted in duplicate with antibodies against angiogenic factors were blocked with blocking buffer and then were incubated overnight with CM. Next, membranes were washed with wash buffer, incubated with biotin-conjugated antibodies against proangiogenic factors, washed with wash buffer, and incubated with horseradish peroxidase–conjugated streptavidin. The signals on the membranes were detected by chemiluminescence. Membranes, blocking and wash buffer, and antibodies against proangiogenic factors were all provided with the kit. The intensity of protein signal (two spots for each protein) was compared with the relative positive signals by densitometric analysis.

Here's an excellent video that provides more detail on Antibody Array Kit capabililities:
I will continue to post information on new addition with related pubs and data.

Monday, August 12, 2013

A Method for 3D Immunostaining and Optical Imaging of the Mouse Brain

Demonstrated in Neural Progenitor Cells

We have proven and effective tools for delivering siRNA, shRNA and miRNA for gene expression analysis and measuring apoptosis in vivo.

We are pleased to introduce a new application for one of our neural progenitor markers. In this study our Chicken Nestin Antibody is used to stain these progenitors in dissected mouse brains: Gleave JA, Lerch JP, Henkelman RM, Nieman BJ (2013) A Method for 3D Immunostaining and Optical Imaging of the Mouse Brain Demonstrated in Neural Progenitor Cells. PLoS ONE 8(8): e72039. doi:10.1371/journal.pone.0072039. 3D antibody staining of adult mouse brains: 1% PFA perfused adult mouse brains were removed from the skull and then divided by removing the cerebellum and separating the hemispheres if desired. The samples were immediately dehydrated in a gradient of methanol solutions to 100% methanol over the course of one day. Immediate dehydration is important to preserve the cellular morphology of the lightly-fixed brain. The samples then underwent freeze/thaw (one hour at –80°C/one hour at room temperature) four times. The samples were rehydrated in a gradient of methanol solutions to PBS over the course of one day. The samples were mildly digested for 5 minutes using 10 mg/ml of Proteinase K (Promega, WI, USA) and then thoroughly washed to remove any residual Proteinase K. Antigen retrieval was performed using a Histos5 histology microwave (Milestone, MI, USA) in 0.01 M sodium citrate buffer pH 6 on an antigen retrieval program (0–90°C 11min, 90–98°C 3min, 98°C 10min). The samples were cooled to RT and subsequently washed in PBS to remove excess buffer. From this point, all incubations were performed in the Histos5 histology microwave. The samples were incubated at 37°C for 48 hours in primary antibody diluted in 5% normal goat serum (Jackson ImmunoResearch, PA, USA), 5% dimethylsulfoxide (DMSO, Fisher Scientific, Ontario, Canada), and 0.01% Triton X-100 (Bioshop Canada, Ontario, Canada). The samples were then washed at 37°C for 48 hours in 5% serum, 0.01% Triton X-100, replacing the solution once after 24 hours. Following this, the samples were incubated for 72 hours in secondary antibody diluted in 5% serum, 5% DMSO, and 0.01% Triton X-100. Finally, the samples were washed at 37°C for 48 hours in 0.01% Triton X-100, replacing the solution once after 24 hours.

Staining of single hemisphere brain samples using the described protocol for 3D imaging included the following: doublecortin (n = 4), GFAP (n = 4), nestin (n = 3), double stain of doublecortin and nestin (n = 1). Doublecortin was also used to stain a full mouse brain excluding the cerebellum (n = 2). Control samples were generated using pre-absorbed primary antibodies when possible by incubating the primary antibody with a blocking peptide for 30 minutes at room temperature prior to staining. If no peptide for the primary antibody was available, the staining procedure was performed in the control except that the primary antibody was omitted during the appropriate step.

Staining was performed with the following antibodies: rabbit anti-doublecortin (Abcam), goat anti-doublecortin and peptide (Santa Cruz Biotechnology) rabbit anti-GFAP (Dako), chicken anti-nestin (Neuromics), goat anti-rabbit alexafluor 546 (Invitrogen), goat anti-rabbit alexafluor 488 (Invitrogen), goat anti-rabbit alexafluor 647 (Invitrogen), goat anti-chicken alexafluor 546 (Invitrogen), goat anti-chicken alexafluor 488 (Invitrogen), donkey anti-goat alexafluor 546 (Invitrogen), donkey anti-goat alexafluor 488 (Invitrogen).


Images: Validation of 3D nestin staining. Cryosections cut from a stained brain hemisphere show nestin-positive cells (red; arrows, A). Some additional punctate fluorescence was present (arrowheads, A). The sections were re-probed with a secondary antibody (green; A) to highlight primary antibody left unbound by secondary antibody. No evidence of primary antibody left unbound by secondary antibody was found. Sections were also re-stained with both primary and secondary antibodies (B) to highlight primary-antibody antigen sites unoccupied. There is overlap of the original 3D nestin stain with the 2D re-stain (arrowheads). LV = Lateral ventricle, scalebars = 100 µm. doi:10.1371/journal.pone.0072039.g003

The best news of all: This staining method is simple, using a combination of heat, time and specimen preparation procedures readily available, so that it can be easily implemented without the need for specialized equipment, making it accessible to most laboratories.