Neuron-Glial Antibodies Hit the Mark!

Neuromics' Neuron-Glial Markers offerings include:

Glial-Astrocyte Markers	Neural Progenitor Markers
Neuron/Synapse Markers	Oligodendrocyte, Oligodendroglial Oligodendrocyte Lineage Markers
Schwann Cell or PNS Neuronal Markers -Peripheral Nervous System (PNS) Related

They are widely used and frequently published. Customers have also shared data which is included in product descriptions. Here is a listing of the most recent pubs:
Wiebke Kallenborn-Gerhardt, Katrin Schröder, Domenico Del Turco, Ruirui Lu, Katharina Kynast, Judith Kosowski, Ellen Niederberger, Ajay M. Shah, Ralf P. Brandes, Gerd Geisslinger, and Achim Schmidtko. NADPH Oxidase-4 Maintains Neuropathic Pain after Peripheral Nerve Injury. The Journal of Neuroscience, 25 July 2012, 32(30): 10136-10145; doi: 10.1523/​JNEUROSCI.6227-11.2012.

Images: MPZ and PMP22 expression in the sciatic nerve of WT and Nox4−/− mice after SNI. A, Western blot analysis of the myelin-specific proteins MPZ and PMP22 in the day 14 SNI sciatic nerve (proximal nerve stump) and the uninjured control sciatic nerve. GAPDH was used as loading control. Note that MPZ and PMP22 protein expression is significantly decreased after SNI in WT mice but not in Nox4−/− mice. n = 3 mice per group. Data are presented as mean ± SEM (*p < 0.05). B, Immunostaining of the day 14 SNI sciatic nerve shows increased MPZ immunoreactivity in Nox4−/− mice compared with WT mice, whereas immunoreactivity of the neuronal marker NF200 is similar in both genotypes. Scale bar, 10 μm.

Maria Maddalena Valente, Valeria Bortolotto, Bruna Cuccurazzu, Federica Ubezio, Vasco Meneghini, Maria Teresa Francese, Pier Luigi Canonico, Mariagrazia Grilli.Alpha2delta ligands act as positive modulators of adult hippocampal neurogenesis andprevent depressive-like behavior induced by chronic restraint stress. Molecular Pharmacology Fast Forward Published on May 9, 2012 as doi:10.1124/mol.112.077636...chicken anti-nestin polyclonal (1:4,000, Neuromics, Edina, Minnesota)...

Frances Y. Cheng, Xi Huang, Anuraag Sarangi, Tatiana Ketova, Michael K. Cooper, Ying Litingtung, Chin Chiang. Widespread Contribution of Gdf7 Lineage to Cerebellar Cell Types and Implications for Hedgehog-Driven Medulloblastoma Formation. PLoS ONE 7(4): e35541. doi:10.1371/journal.pone.0035541...rabbit anti-GFAP (Neuromics, 1:500), mouse anti-GFAP (Neuromics, 1:200)...

Juan M Jimenez-Andrade and Patrick W Mantyh. Sensory and sympathetic nerve fibers undergo sprouting and neuroma formation in the painful arthritic joint of geriatric mice. Arthritis Research & Therapy 2012, 14:R101...to label primary afferent sensory nerve fibers, an antibody against neurofilament 200 kDa (NF200, chicken anti neurofilament 200 kDa; NF200, 1:5000; Neuromics; catalog #CH22104)...

I will be posting new developments.

Converting Human Pluripotent Stem Cells into Nociceptors

Methods for differentiating induced pluripotent stem cells (iPSCs) into specific cell types are a requirement for converting the "promise of iPSCs" into reality. The knowledge derived from this research can be leveraged for high throughput  Drug Discovery and ultimately the development of therapies. I am excited to highlight results recently published by Dr. Lorenz Studer and his team at Memorial Sloan-Ketterling: Stuart M Chambers, Yuchen Qi, Yvonne Mica, Gabsang Lee, Xin-Jun Zhang, Lei Niu, James Bilsland, Lishuang Cao, Edward Stevens, Paul Whiting, Song-Hai Shi, Lorenz Studer. Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors. Nature Biotechnology 30, 715–720 (2012) doi:10.1038/nbt.2249.

Abstract: Considerable progress has been made in identifying signaling pathways that direct the differentiation of human pluripotent stem cells (hPSCs) into specialized cell types, including neurons. However, differentiation of hPSCs with extrinsic factors is a slow, step-wise process, mimicking the protracted timing of human development. Using a small-molecule screen, we identified a combination of five small-molecule pathway inhibitors that yield hPSC-derived neurons at >75% efficiency within 10 d of differentiation. The resulting neurons express canonical markers and functional properties of human nociceptors, including tetrodotoxin (TTX)-resistant, SCN10A-dependent sodium currents and response to nociceptive stimuli such as ATP and capsaicin. Neuronal fate acquisition occurs about threefold faster than during in vivo development(1), suggesting that use of small-molecule pathway inhibitors could become a general strategy for accelerating developmental timing in vitro. The quick and high-efficiency derivation of nociceptors offers unprecedented access to this medically relevant cell type for studies of human pain

Figure – LSB3i Differentiation model. Early LSB inhibits trophectoderm, mesendoderm, and non-neural ectoderm cell fates yielding neuroectoderm. CHIR99021, SU5402 and DAPT induce and accelerate neural crest stem cell identity by day 8 and promote rapid differentiation of the neural crest stem cells to nociceptors expressing peptidergic markers by day 10.

Note: Neuromics' TRPV1 Antibody was used as a marker for mature nociceptors.

Check out Supplementary Data for more. I will continue to post links to methods here and @ Neuromics' Stem Cell Research Reagents.

mGluR Antibodies in Action

Metabotropic glutamate receptors (mGluRs) are involved in normal brain function and can be perturbed in many neuropathologic conditions. This makes mGluR Markers important tools for Neuroscience Research.

Neuromics has a comphrensive catalog of these markers. They are widely used and frequently reference in pubs-Neuromics' mGluR customer publications. I get excited when a mGluR we recently manufactured is referenced in a publication: Hoon Shim, Chih-Ting Wang, Yen-Lin Chen, Viet Q. Chau, Kevin G. Fu, Jianqi Yang, A. Rory McQuiston, Rory A. Fisher, and Ching-Kang Chen. Defective Retinal Depolarizing Bipolar Cells (DBCs) in Regulators of G-protein Signaling (RGS) 7 and 11 Double Null Mice. JBC Papers in Press. Published on February 27, 2012 as Manuscript M112.345751. The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M112.345751...Animals were sacrificed by CO2 inhalation and the eyeballs were immediately enucleated. After removal of cornea and lens the resulting eyecups were immersionfixed in 4% paraformaldehyde in 1X PBS at room temperature for 15 minutes. This short fixation time ensured good mGluR6 and Gb5 signals at the OPL. After cryoprotection in 30% sucrose in 1X PBS, the eyecups were embedded in TBS (Richard Allan Scientific, Kalamazoo, MI), sectioned at 20μm thickness, and stained...

Images: Absence of Gb5 immunoreactivity in mGluR6-containing puncta in retinas of RGS7 and RGS11 double knockout (711dKO) mice. Retinal sections from wild-type control (WT) and 711dKO (DKO) mice stained for metabotropic glutamate receptor 6 (mGluR6, 1:200) and Gb5 (CT215, 1:250) demonstrates superimposed signals in the WT but absence of Gb5 signal in the DKO. Scale bar equals 10μm.

I will continue to post new developments reagrding this important category of Neurotransmission Research Antibodies.

FLICA™ in vitro Caspase Kits for Apoptosis Assays

Great solution for measuring neurotoxicity

Here's a recent pub confirming the capabilities of our FLICA™ in vitro Caspase Apoptosis Detection Kits: Minghai Zhou, Gregory Ottenberg, Gian Franco Sferrazza, and Corinne Ida Lasmézas Highly neurotoxic monomeric α-helical prion protein. PNAS 2012 109 (8) 3113-3118; published ahead of print February 7, 2012, doi:10.1073/pnas.1118090109. ...PFA and stained for caspases using FLICA polycaspase assay (Neuromics) according to the manufacturer's instructions. Caspases...assayed on live cells using the caspase- specific FLICA assay (Neuromics). Cells were photographed using a Nikon inverted epifluorescence...

Images: FLICA 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.

I would like to underscore that we have solutions to cover most your needs for Apoptosis and Cytoxicity Assays. I will continue to post updates on customer data and publications.

eSC Derived human Mesenchymal Progenitor Pubs

We have added hMPro^TM Mesenchymal Progenitors to our Stem Cell Research Reagents category. These progenitors coupled with our UBC derived Human Mesenchymal Stem Cells give more options to basic and drug discovery researchers. Since they serve as potent research tools, it is a strategy of ours to continue to broaden our offerings in this products category.

Publications matter when assessing the capabilities of bio-reagents. I would like to share several that prove the utility of these cells:  David L. Simpson, Nolan L. Boyd, Sunjay Kaushal, Steve L. Stice, Samuel C. Dudley Jr. Use of human embryonic stem cell derived-mesenchymal cells for cardiac repair. Biotechnology and Bioengineering Volume 109, Issue 1, pages 274–283, January 2012. DOI: 10.1002/bit.23301

Nolan L. Boyd, Ph.D., Kelly R. Robbins, Ph.D., Sujoy K. Dhara, D.V.M., Ph.D., Franklin D. West, Ph.D., and Steven L. Stice, Ph.D. Human Embryonic Stem Cell–Derived Mesoderm-like Epithelium Transitions to Mesenchymal Progenitor Cells. Tissue Eng Part A. 2009 August; 15(8): 1897–1907. Published online 2009 January 15. doi: 10.1089/ten.tea.2008.0351.

Images: (A) Phase contrast image of hMPro™ mesenchymal progenitor cells in culture. (B) Confocal image of hMPro™ cells stained for the early smooth muscle marker (αSMA;green), F-actin (red) and nuclei (blue). Exposure to 10ng/mL of transforming growth factor beta 1 (TGF-β1) for 12 days induces expression of alpha smooth muscle actin (αSMA) in WA09-derived mesenchymal progenitor cells, suggesting their ability to differentiate along the smooth muscle lineage.

These cells have the ability to:

•Form adherent monolayers – ideal for 96- and 384-well formats for high throughput and high content cell-based assays.
•Differentiate into a wide variety of mesenchymal subtypes, including osteogenic and chondrogenic lineages (but not adipogenic)– ideal for drug discovery for a wide variety of biological targets and basic research in bone, cartilage, metabolic and immunological diseases.
•Passage up to 10X.

I will keep you posted on new applications.

TLR3s and the Synaptic Transmission of Itch

Synaptic Transmission Markers Trifecta

Itch, also known as pruritus, is a common, intractable symptom of several skin diseases, such as atopic dermatitis and xerosis. This chronic condition erodes quality of life.

The authors of this publication have made an important discovery that could prove a target for treating chronic itch. They also used 3 of our markers to confirm this discovery-guinea pig anti-TRPV1, guinea pig anti-SP antibody and rabbit anti-CGRP antibodies: Tong Liu, Temugin Berta, Zhen-Zhong Xu,Chul-Kyu and Ru-Rong Ji. TLR3 deficiency impairs spinal cord synaptic transmission, central sensitization, and pruritus in mice. J Clin Invest. 2012 June 1; 122(6): 2195–2207. Published online 2012 May 8. doi:10.1172/JCI45414.

Highlights: Scratching behaviors induced by histamine-dependent and -independent pruritogens are markedly reduced in mice lacking the Tlr3 gene. TLR3 is expressed mainly by small-sized primary sensory neurons in dorsal root ganglions (DRGs) that coexpress the itch signaling pathway components transient receptor potential subtype V1 and gastrin-releasing peptide. Ttreatment with a TLR3 agonist induces inward currents and action potentials in DRG neurons and elicited scratching in WT mice but not Tlr3–/– mice. Furthermore, excitatory synaptic transmission in spinal cord slices and long-term potentiation in the intact spinal cord were impaired in Tlr3–/– mice but not Tlr7–/– mice. Consequently, central sensitization–driven pain hypersensitivity, but not acute pain, was impaired in Tlr3–/– mice. In addition, TLR3 knockdown in DRGs also attenuated pruritus in WT mice. Finally, chronic itch in a dry skin condition was substantially reduced in Tlr3–/– mice. This demonstrates a critical role of TLR3 in regulating sensory neuronal excitability, spinal cord synaptic transmission, and central sensitization.

Images: Impaired scratching behaviors and reduced c-Fos expression in the spinal cords in Tlr3–/– mice. (A and B) Scratches in every 5 minutes (left) and 0–30 minutes (right) induced by intradermal injection of 50 μl compound 48/80 (100 μg) and CQ (200 μg). Note a reduction of both histaminergic (compound 48/80) and nonhistaminergic (CQ) itch in Tlr3–/– mice. *P < 0.05, Student’s t test; n = 11–13 mice for each group. Mean ± SEM. Two-way repeated-measures ANOVA analysis also shows a significant difference in the time course of compound 48/80– and CQ-induced scratching between the 2 groups (P < 0.05). (C) c-Fos–like immunoreactivity in the dorsal horn of the cervical spinal cord in WT and Tlr3–/– mice 2 hours after intradermal injection of compound 48/80 (48/80) or CQ. Right panels show the number of c-Fos–positive neurons in the dorsal horn. Scale bars, 100 μm. *P < 0.05, Student’s t test; n = 4–6 mice. All the data are mean ± SEM.

Images: Expression of TLR3 in a subset of small-sized DRG neurons. (A) Single-cell RT-PCR analysis from dissociated small-sized DRG neurons showing the distinct and overlapped distribution patterns of TLR3 and TLR7 in DRG neurons. The lanes were run on the same gel but were noncontiguous. M, marker; NC, negative control. (B) Single-cell RT-PCR analysis from dissociated small-sized DRG neurons showing colocalization of TLR3 with TPRV1 and GRP. Similar results were obtained from 3 independent experiments in 30 cells collected from different animals. (C) Double immunostaining in DRGs showing co-colocalization of TLR3 and GRP. Red and yellow arrows indicate GRP+ only and double-labeled neurons, respectively. Scale bars: 50 μm. (D) Cell size distribution frequency of TLR3+ and GRP+ neurons. (E) Double immunostaining in cultured DRG neurons showing co-colocalization of TLR3 with TRPV1 but not with NF200. Green arrows indicate NF200+ or TRPV1+ neurons, red arrows indicate TLR3+ neurons, and yellow allows indicate double-labeled neurons. Scale bars: 50 μm. (F) A Venn diagram showing the relationship of TLR3+, GRP+, and TRPV1+ populations in a DRG. Note that all TLR3+ cells also express GRP and TRPV1.

Nociceptive DRG neurons are involved in itch. TRPV1 and CGRP are indispensible for itch sensation. Given that all TRL3+ cells express these proteins, TRL3 could be a viable target for treating itch.