Neuromics New Neuronal Markers

Best of the Best
We are recognized for having some of the best Neuronal-Glial Antibodies in the business. We are pleased to announce the addition of these:

CH22122 – Tyrosine Hydroxylase

GT22101 - GFP

GT22102 – MAP2

GT22103 - Vimentin

MO22186 – Tyrosine Hydroxylase

RA22135 – Tyrosine Hydroxylase

Immunofluorescent analysis of rat brainstem section stained with GT22102, MAP2 dilution 1:2,000 in red, and costained with mouse mAb to MO22121, MBP dilution 1:5,000, in green. Following transcardial perfusion of rat with 4% paraformaldehyde, brain was postfixed for 24 hours, cut to 45μM, and free-floating sections were stained with the above antibodies.

If you have questions on these or any of our solutions, not hesitate to contact me directly-pshuster@neuromics.com or 612-801-1007,

Quality, High Titer Neuronal Markers

You Need Them; We got Them

We are recognized for both the number and quality of Neuronal Markers. Check out our many testimonials from satisfied customers.

Here's a sampling of recent publications.

Elisabet Garcia-Pino, Nikodemus Gessele and Ursula Koch. Enhanced Excitatory Connectivity and Disturbed Sound Processing in the Auditory Brainstem of Fragile X Mice. Journal of Neuroscience 3 July 2017, 2310-16; doi.org/10.1523/JNEUROSCI.2310-16.2017. ...chicken α-MAP2 (Neuromics; dilution 1:1000)...
Anna Lisa Gündner Claas Aiko Meyera, Stefan Aigner, Klaus Christensen, Christoph Patsch, Ravi Jagasia, Karlheinz Baumann, Marc Burcin. Generation of a homozygous GBA deletion human embryonic stem cell line. Stem Cell Research. Available online 11 July 2017. https://doi.org/10.1016/j.scr.2017.07.009 ...Neuronal differentiation marker, Ch-MAP2, 1:1000, Neuromics # CH22103 RRID:AB_2314763 ....Immunocytochemistry. Cells were cultured on PO/LAM coated 96-well imaging microplates (Falcon) and fixed with 4% paraformaldehyde (15 min, room temperature). Fixed cells were permeabilized with 0.2% Triton (Sigma) in phosphate-buffered saline (PBS) for 30 min and blocked for 1 h with PBS + 5% donkey serum (Merck Millipore). Primary antibodies (in PBS) were incubated overnight at 4 °C followed by three PBS washing steps. Secondary antibodies were incubated for 2 h at room temperature. Confocal images were acquired using a Leica TCS SP5 microscope (Leica Microsystems)....
Hayk Harutyunyan, Svetlana Sharoyan, Alvard Antonyan, Sona Mardanyan. Herb Preparations Improve the Viability of Hippocampal Cells Suppressed by Amyloid Beta (1-42) Peptide. World Journal of Pharmaceutical Sciences. 2017. ISSN (Online): 2321-3086......Antibodies for immunocytochemistry were purchased from Neuromics (USA)[....]Immunocytochemistry: Hippocampal cells were cultured in Poly-D-Lysine coated Nunc EasY Flasks. On the 4th day, the adhered cells were removed by trypsinisation (0.05% trypsin, 0.5 mM EDTA, pH 8.0). Cells were fixed on microscope slide by 3.7 % paraformaldehyde in PBS followed by methanol treatment. To block a nonspecific antibody binding, samples were pre-treated with goat serum (Sigma) for 30 minutes. To determine the types of cells, constituting the obtained cell culture, the different aliquots of the culture were incubated for 60 min at room temperature with the primary antibodies against rabbit anti-rat Neurofilament (NF), chicken anti-rat GFAP and Nestin...

Images: Cells grown from adult rat brainLarge cell in middle is stained with mouse monoclonal to NF-L clone DA2 (green). Another type of neuronal lineage cell was stained with rabbit polyclonal to alpha-internexin (red). These cells were mitotic but had several characteristics of neurons. Rat spinal cord homogenate showing the major intermediate filament proteins of the nervous system (lane 1). The remaining lanes show blots of this material stainted with various antibodies including: NF-H, NF-M,, NF-L,, NF66 and GFAP 

We will continue to post the latest and greatest news.

Neuropathy Research Solutions

New Publications and Past Postings

Here're are several key postings on Neuropathy and Neuropathic Pain:

• Cytokines and Neuropathic Pain
• Epidermal Nerve Fibers in Neuropathic Pain Model
• Pain Research and Gene Expression Analysis

Here're some "hot of the press" publications referencing use of Neuron-Glial Markers: 

Bethany L. Johnson-Kerner, Faizzan S. Ahmad, Alejandro Garcia Diaz, J. Palmer Greene, Steven J. Gray, R. Jude Samulski, Wendy K. Chung, Rudy Van Coster, Paul Maertens, Scott A. Noggle, Christopher E. Henderson and Hynek Wichterle. Intermediate filament protein accumulation in motor neurons derived from giant axonal neuropathy iPSCs rescued by restoration of gigaxonin. Hum. Mol. Genet. (2014) doi: 10.1093/hmg/ddu556. First published online: November 4, 2014.
...MAP2 (1:2,000, Neuromics, CH22103), NF-H (1:2000, Neuromics, CH22104), NF-L (1:200, Neuromics, MO22104)...

Images: Cells grown from adult rat brain. Large cell in middle is stained with mouse monoclonal to NF-L clone DA2 (green). Another type of neuronal lineage cell was stained with rabbit polyclonal to alpha-internexin (red). These cells were mitotic but had several characteristics of neurons. Rat spinal cord homogenate showing the major intermediate filament proteins of the nervous system (lane 1). The remaining lanes show blots of this material stainted with various antibodies including: NF-H, NF-M,, NF-L,, NF66 and GFAP 

Jianfei Guo, Xudong Fu, Xia Cui, Minhua Fan. Contributions of purinergic P2X3 receptors within the midbrain periaqueductal gray to diabetes-induced neuropathic pain. The Journal of Physiological Sciences November 2014.
...An equal volume of total and membrane samples was applied to SDS-PAGE. Membranes were incubated with the rabbit anti-P2X 3 primary antibody (1:1000, Neuromics, Edina, MN, USA) and goat anti-rabbit secondary antibody (1:200, Neuromics, Edina, MN, USA).

Neuropathy Research Solutions Include:

Antibodies Antibodies by Category

Apoptosis Research Reagents Detection kits, antibodies and proteins by category

ELISA Solutions ELISA kits, buffers, diluents and stabilizers by Category

Labeling Kits and Reagents Labeling kits, Cloning-Purification kits, Fluoro-tracers, i-Brite+, GFP and Related Markers

Cells, Cell Culturing Systems and Cell Based Assay Reagents Cells, Petaka Cell Culturing Systems, Biolumonics Kits, Media, 3-D Scaffolds and Related Reagents Designed to Optimize Cell Based Assays

Primary Neurons and Astrocytes Primary human, rat and mouse neurons and astro-glia by category

Proteins, Growth/Differentiation Factors and Small Molecules Recombinant/natural proteins plus small molecule/peptide agonists, antogonists, inihibitors and ligands

Stem Cell Research Reagents Cell, Media, Markers and Growth/Differentiation Factors

Transfection Reagents Transfection kits and reagents by Category

Potents Tools for Neuroscience Based Toxicology Assays

Neuromics' Offers Best in Class Cell and Markers

I am always on the hunt for proof that are tools work in the many different applications required by Researchers Studying Neurotoxicology. Success is confirmed to us through Customer Data/Pubs and Testimonials.

I would like to feature here some examples:

• Neuron-Glial-Astrocyte-Progentitor Markers-Bin Xing, Adam D. Bachstetter, Linda J. Van Eldik. Inhibition of Neuronal p38α, but not p38β MAPK, Provides Neuroprotection Against Three Different Neurotoxic Insults. Journal of Molecular Neuroscience. July 2014...Cells were fixed with 3.7 % formaldehyde containing 0.1 % Triton X-100 in PBS for 10 min at room temperature. After washing three times with PBS, the coverslips were incubated with blocking buffer (PBS containing 6 % goat serum, 3 % bovine serum albumin (fraction V), 0.1 % Triton X-100) for 30 min at room temperature. Primary chicken anti-MAP2 antibody (1:1,000, Neuromics, Cat. no. CH22103) was diluted in blocking buffer and incubated with the cells at room tem- perature for 2 h. For detection of MAP2 staining, the cells were incubated with secondary biotin SP-conjugated goat antichicken antibody (1:1,000, Jackson ImmunoResearch) for 1 h, followed by streptavidin Alexa Fluor® fluorescent 488 (1:1,000, Invitrogen) incubation in blocking buffer at room temperature for 1 h. Wide field fluorescent photomicro- graphs were obtained using a Nikon Eclipse Ti microscope with an Axiocam MRc5 digital camera (Carl Zeiss)...

Figures: Neurons stained with Neuromics' MAP2 antibody to determine Neurite Damage.

• Primary Neurons and Astrocytes-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 ...Primary hN2™ Human Neurons (Neuromics) were grown in hN2™ human neuron culture media (Neuromics)..
• Xiugong Gao, Hsiuling Lin, Radharaman Ray, Prabhati Ray. Toxicogenomic Studies of Human Neural Cells Following Exposure to Organophosphorus Chemical Warfare Nerve Agent VX. Neurochemical Research. February 2013. ...Human hN2 neurons were obtained from Neuromics...

We guarantee results. If you would like to learn more, please contact me directly at pshuster@neuromics.com or direct phone line: 612-801-1007. Thank you.

MAP-2-A Versatile Neuron Marker

Neuromics is a leader in providing Neuron-Glial Markers for Neuroscientists.

We are constantly on the search for publications that reference use of these markers in unique applications. In this posting I would like to share a publication where researchers used on of our MAP-2 antibodies to stain medial superior olive (MSO) neurons. Baumann Veronika, Lehnert Simon, Leibold Christian, Koch Ursula. Tonotopic Organization of the Hyperpolarization-activated Current (Ih) in the Mammalian Medial Superior Olive. Front. Neural Circuits 7:117. doi: 10.3389/fncir.2013.00117.
 ...Following recording, slices were fixed in 4% paraformaldehyde for 30 min. After extensive washing in phosphate-buffered saline (PBS) slices were exposed to blocking buffer (0.5% trition X-100/0.1% saponin/1% BSA in PBS) followed by incubation with the primary antibody (chicken anti-microtubule-associated protein 2, MAP2, 1:1000, Neuromics) in blocking buffer. Slices were then rinsed in washing buffer (0.5% Trition X-100/0.1% saponin in PBS) and immunoreactivity was visualized by incubating the slices with the Cy3-conjugated secondary antibody raised in donkey (1:300; Dianova). Finally, slices were washed and mounted on slides with vectashield mounting reagent (Vector Laboratories, USA)...

Here the MAP-2 antibody is used to help identify the dorsal, medial and ventral portion of the MSO of p18 and p22 gerbils.

Figure . Ih varies systematically along the dorsoventral axis. (A) A brain slice containing the MSO with Alexa-488-filled neurons (green) verifies the distribution of the patched neurons along the dorsoventral axis (red: MAP-2). (B) Pharmacologically isolated Ih current traces were elicited by depolarizing and hyperpolarizing voltage steps from −60.5 mV to potentials between −40.5 mV and −120.5 mV for 1 s in 5 mV step increment and then to −100.5 mV for 0.5 s to elicit the tail current to determine the voltage dependence of Ih activation. Current traces are representative for the dorsal, the intermediate and the ventral part of the MSO. (C) I-V relationships of steady-state (red arrow in B) Ih density for ventral (n = 15), intermediate (n = 12) and dorsal (n = 18) neurons emphasize that Ih density amplitudes are smallest in dorsal neurons and largest in ventral neurons (C1). Ih density amplitudes for a voltage step to −110.5 mV (C2). (D) Weighted activation time constants at −110.5 mV (D1). The weighted activation time constants are voltage dependent and largest in the dorsal part of the MSO (D2). (E) The voltage-dependence of Ih activation was measured from the tail current 20 ms after the end of the voltage steps (red arrow) (E1). Values were fitted with a Boltzmann function to obtain the half-maximal activation voltage. In dorsal neurons the Ih activation curve is shifted to more negative voltages (E2). Half-maximal activation voltage was measured in each experiment and averaged (E3). Black symbols: dorsal neurons; gray symbols: intermediate neurons; white symbols: ventral neurons. **P < 0.01, ***P < 0.001, single-factor ANOVA test followed by a Scheffe's post-hoc test.

I will continue to post interesting applications using our Neuron-Glial Markers.

Neuron-Glial Cultures-Setting a Higher Bar!

Improved Methods for Long Term, High Denisty Cultures

 

Dr. Randen Patterson and his team at UC Davis have developed new culturing techniques using our e18 Rat Primary Hippocampal Neurons. They have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes–particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons. This is good news indeed: Todd GK, Boosalis CA, Burzycki AA, Steinman MQ, Hester LD, et al. (2013) Towards Neuronal Organoids: A Method for Long-Term Culturing of High-Density Hippocampal Neurons. PLoS ONE 8(4): e58996. doi:10.1371/journal.pone.0058996.

 

Protocol Highlights: 
Substrate Preparation
1. On the day of plating, prepare 25 mm coverslips by removing them from 70% ethanol storage solution and propping them up at an angle in each well of a 6-well culture plate to allow drying. [No more than 5 plates (30 coverslips) should be dried simultaneously for 15–25 minutes in culture hood to avoid over-drying.]
2. Once dry, shake slips down flat into their respective wells and coat with 1 mL 0.1% poly-D-lysine, taking care to form a liquid meniscus on each slip. Carefully transfer coverslips into incubator, taking care to preserve meniscus.
3. Incubate for 1 hr at 37°C. [Keeping poly-D-lysine meniscus on top of coverslip is important; this serves to avoid poly-D-lysine coating under coverslip surface that may lead to problematic flotation of coverslip.] After incubation, remove poly-D-lysine and rinse each coverslip three times with 2 mL sterile deionized water. Take care to ensure coverslips do not completely dry at any point during the rinse. After the third and final rinse, leave coverslips in 2 mL sterile deionized water for at least 1 hr. Remove water just before plating, again, make sure to avoid over-drying. [This critical step requires attention. Take care to aspirate off all sterile water remaining from the final rinse, but also use caution as not to over-dry the coverslips. Ultimately, the coverslip must be mostly dry as to allow for the meniscus formation during plating (Fig. 1, Step 12), whereas over-drying can result in the neurons peeling off the glass coverslips days to weeks after plating.]

Fig 1: Neuron-Glial Culturing Steps
Preparation of Isolated Neurons (Numbers in Fig. 1 correspond to numbers below).
1. Store tissue at 4°C until ready to use. If dissecting your own cultures, upon isolation of the tissue, store in an appropriate storage media.
2. When ready to plate, make 2 mL of enzymatic solution without B27. In our case, we used Hibernate E-Ca, containing 4 mg (2 mg/mL) of papain. If making your own solution, use a commercially available papain dissociation kit. Make sure to sterile filter solution with 0.2 micron filter after adding papain if source of enzyme is not sterile.
3. Remove the storage media from the dissected tissue and transfer into sterile 15 mL screw-cap tube; be careful not to disturb or remove tissue from original tube. Save the storage media, do not discard.
4. Add 2 mL of media made in Step 2 to tissue (in our case, Hibernate E-Ca containing 2 mg/mL of papain). Incubate for 35 min at 37°C. [Be sure to add Hibernate E-Ca containing papain slowly as to avoid disturbing tissue.]
5. Remove enzymatic solution from tissue, again, take care not to disturb or remove tissue. Add back 1 mL of storage media saved in 15 mL tube.
6. Using a 1 mL pipettor with a sterile plastic pipette tip (tissue can adhere to glass pipettes), aspirate the tissue with the medium into the pipette and immediately dispense contents back into same container. Take care not to create bubbles. [This is another critical step that requires attention. Take care to make sure pipette tip remains in a stable position (as shown in Fig. 1, Step 6). Maintain slow, steady speed when both drawing in and re-dispensing media containing tissue.]
7. Repeat this trituration step 10–12 times or until most all the tissue is dissociated and the cells are dispersed. [Under close examination cell dispersion is highly visible. Stop pipetting immediately upon cell dispersion.]
8. Slowly transfer contents of the tissue tube into a new sterile 15 mL screw-cap tube.
9. Use the remainder of storage medium saved in Step 3 and rinse the interior of the tissue tube before adding it to the sterile 15 mL screw cap tube containing dispersed cells from step 7. [This step helps ensure minimal wastage, as any remaining cells should be saved with this extra rinse.]
10. Spin dispersed cells at 1,100 rpm (200Xg) for 1 min.
11. Discard the supernatant while being careful not to remove any of the cells from cell pellet.
12. Flick tube a few times to loosen the cell pellet. Re-suspend pellet in 2.4 mL of pre-warmed B27/Neurobasal/0.5 mM glutamine medium. Re-suspend by gently pipetting up and down. For E18 Hippocampus, medium includes 25 µM glutamate.
13. Plate cells within a meniscus (approx. 10 mm diameter) at a minimum of 40 µL per 25 mm coverslip. Take care not to disturb meniscus. [Periodically pipette up and down throughout plating process (no more than once every plate per 6 coverslips) to help maintain equal cell density. Again, plating with meniscus formation is critical.]
14. Incubate plated cells at 37°C with 5% CO2 and/or 9% or 20% oxygen for 1 hr.
15. Add 1.5 mL per well of pre-warmed 1:1 ACM/NbActiv4. [Slow and steady media addition rate and proper pipette position are necessary for successful plating density consistency. Position pipette tip at 45° angle along middle of 6-well interior sidewalls, dispense 1.5 mL as slowly and steadily as possible (see Steps 1–14).]
16. Incubate cells at 37°C with 5% CO2 and/or 9% or 20% oxygen.
17. Add Cytosine β-D-arabinofuranoside (Ara-C) to a final concentration of 5 µM, 5–6 days after plating to curb glial proliferation. [Remove 1/3 of media from each well and replace with equal volume containing final concentration of Ara-C]
18. After 4 days or longer, neurons are well differentiated. If further culture is desired, change 1/3 of medium with fresh, pre-warmed 1:1 ACM/NbActiv4 every 7–8 days.

Images: 40X Confocal Images of 30 DIV Hippocampal Cultures. Immunofluorescence detection of MAP-2 (green) and GFAP (red) in 30 DIV (A–C) cultured E18 hippocampal cells using a 40X objective. These images (A and B) clearly depict the intimate physical contact between glia processes and dendritic arbors. Under closer examination (CI and CII), it is clear that the dendrites have grown both bellow (blue arrows) and above (white arrows) glial processes, forming a highly interconnected three-dimensional network by 30 DIV. doi:10.1371/journal.pone.0058996.g004.
All Primary Neuron Assay Customer Publications

 

Related Content:

• Primary Neurons and Astrocytes-Primary human, rat and mouse neurons and glia/astrocytes.
• Neuronal-Glial Markers-Astrocytes, Glia, Microglia, Olidogodendrocytes, Progenitors and Schwann Cell Markers
• Cells, Cell Culturing Systems and Cell Based Assay Reagents-Cells, Petaka Cell Culturing Systems, Biolumonics Kits, Media, 3-D Scaffolds and Related Reagents Designed to Optimize Cell Based Assays

If you have any questions on optimizing your cell cultures, do not hesitate to contact me @ pshuster@neuromics.com or 612-801-1007

Stress, Depression and Alpha2delta Ligands

Implications for treating Stress Related Depression.

Stress related disorders like Post Traumatic Stress Syndrome (PTSD), Major Depressive Disorder (MDD) and Generalized Anxiety Disorders (GAD) dysregulate neurogenesis. This disregulation can lead to disorders like chronic depression. Indeed, stress hurts.

In this study (which includes use of our Neural Progenitor Marker-Nestin), researchers show for the first time that the alpha2delta (α2δ) ligands gabapentin [1- (aminomethyl)cyclohexaneacetic acid; GBP] and pregabalin [S-[+]-3-isobutylGABA or (S)-3- (aminomethyl)-5-methylhexanoic acid; PGB] can produce a concentration-dependent increase in the number of newborn mature and immature neurons generated in vitro from adult hippocampal neural progenitor cells (NPC), and, in parallel, a decrease in the number of undifferentiated precursor cells. These effects were confirmed in vivo, since a significantly increased number of adult generated neurons was observed in the hippocampal region of mice chronically treated with PGB [10 mg/kg, i.p., 21 days] compared to vehicle-treated mice. Moreover, we demonstrated that PGB administration prevented the appearance of depression-like behaviours induced by chronic restraint stress and, in parallel, promoted hippocampal neurogenesis in adult stressed mice. Finally, we provided data suggesting the potential involvement of the α2δ1 subunit and NF-κB signaling pathway in the drug-mediated proneurogenic effects. The new pharmacological activities of α2δ ligands may help explaining their therapeutic activity as add-on therapy in major depression and on depressive symptoms in posttraumatic stress disorder and generalized anxiety disorders. Furthermore these data contribute to the identification of novel molecular pathways which may represent potential targets for pharmacological modulation in depression: 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.

Images: Effect of α2δ ligands on neuronal differentiation and proliferation of hippocampus-derived neural progenitor cells. (A) Representative fluorescence microscopy image of a hippocampal neurosphere immunolabelled for nestin (green) and SRY-related HMG-box gene 2 (Sox-2) (red), markers of undifferentiated NPC. Magnification = X600. Scale bar = 56 μm. (B) After 24 h in absence of growth factors, hippocampal Neural Progenitor Cells (NPC) differentiated giving rise to four different cell populations identified by double Microtubule Associated Protein-2 (MAP-2) and nestin immunolabelling: MAP-2+/nestin- mature neurons, MAP-2+/nestin+, MAP-2-/nestin+ and MAP-2-/nestin- cells. Data are expressed as mean ± S.D. of n=9 experiments, run in triplicates. Gabapentin (GBP) and pregabalin (PGB) promote neuronal differentiation of adult hippocampal NPC. GBP (C-F) and PGB (G-J) significantly increased, in a concentrationdependent manner, the percentage of MAP-2+/nestin- (C, G) and MAP-2+/nestin+ (D, H) cells and decreased the percentage of MAP-2-/nestin- cells (F, J), with no effect on MAP-2-/nestin+ cells (E, I). Data are expressed as mean ± S.D. of n = 3 experiments, run in triplicates. *, p < 0.05; **, p < 0.01; ***, p < 0.001 vs vehicle (Student’s t-test). (K-M) Representative fluorescence microscopy images of MAP-2 immunolabelling (green) in cells derived from hippocampal NPC after 24 h treatment with vehicle (K), 1 nM GBP (L) and 1 nM PGB (M). Nuclei are stained with Draq5(blue). Magnification = X400. Scale bar = 75 μm. (N) Adult hippocampal NPC were treated with vehicle or 1 nM PGB for 6, 24, 48, 72, 96 h and proliferation rate was assessed. PGB had no effect on NPC proliferation, when compared to vehicle. Data, expressed as counts per second (CPS), represent the mean ± S.D. of experiments run in triplicates.

This study is good news. Results demonstrate the new pharmacological activity of α2δ ligands may

potentially explain their efficacy as add-on therapy in MDD, as well as on depressive symptoms
in PTSD and GAD. This knowledge will help the discovery of refined therapies for these debilating disorders.

Primary Neuron Assays for Studying Neurodegeneration

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

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

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

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

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

IF Staining of Human Primary Neurons

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

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

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

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

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

Markers for Medial Superior Olivary Neurons

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

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

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

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

human, rat and mouse neurons and astrocytes.

Fragile-X, Astrocytes and BMC Image of the Month

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

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

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

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

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

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

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

Potent Neuron-Glial Markers

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

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

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

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

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

Related Reagents

• Stem Cell Research Antibodies


• Stem Cell Research Reagents


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