Anti neun antibody

LPS, ATP, BzATP (catalog number B6396), and the antibody anti-α-tubulin (catalog number T6199) were purchased from Sigma-Aldrich (Madrid, Spain). Anti-NeuN antibody (catalog number MAB377) were obtained from Merck Millipore (Tullagreen, Ireland). A438079 (catalog number 2972/10) was obtained from Tocris BioScience (Bristol, United Kingdom). Monoclonal anti Aβ 4–10 residues clone WO2 (catalog number MABN10) was purchased from Merck Chemicals (Madrid, Spain). The commercial antibody for P2X7R (catalog number APR-004) was purchased from Alomone Labs (Jerusalem, Israel). Antibodies against enhanced green fluorescent protein (EGFP) were obtained from Thermo Fisher (catalog number A11122) and AVESLab (catalog number GFP-1020) (Tigard, OR, United States). Red Fluorescent 2 μm diameter microspheres (catalog number F8826) (Life technologies). Antibody against Iba-1 (catalog number 019-19741) was provided from Wako (Richmond, VA, United States). CytoD (catalog number sc-201442) was provided by Santa Cruz (Dallas, TX, United States). Selective P2X7R antagonist GSK 1482160A was provided by GlaxoSmithKline (Madrid, Spain)

TUNEL assays were performed according to the protocol using an in situ cell death assay kit (Roche, Basel, Switzerland). Sections were re-stained with anti-NeuN antibody (1:200; Merck Millipore, Hong Kong, China) for 3 min. Tunel-positive cells were counted in six different microscopic fields from the CA1 region of each section of the hippocampus and the percentage was calculated.

The TUNEL assay was carried out with the In Situ Cell Death Detection Kit (Roche Inc., Indianapolis, IN) following the protocols. Sections were counterstained by Anti-NeuN Antibody (1:200, Merck Millipore) for 3 min, washed with PBS three times, and covered by microscopic glass with Antifade Mounting Medium (Beyotime) for further analyses. TUNEL-positive cells in the hippocampal CA1 region were visualized and counted using a ZEISS HB050 inverted microscope system (Zeiss, Jena, Germany). The density of TUNEL positive cells in CA1 region was calculated by dividing the number of TUNEL-positive cells by the area of CA1 region.

Fifteen animals were anaesthetised with urethane, transurethrally catheterised, and treated with 200 μl of 0.25 IU chondroitinase ABC and heparanase III. Then, five animals each had 10 μl of 250 ng/ml of either CCL21 or FGF7 instilled. Five control animals were catheterised without bladder instillation. After 2 h, all animals were sacrificed and transcardially perfused with 4% paraformaldehyde (VWR, Lutterworth, UK). Spinal cord sections L6–S1 were collected, cryoprotected, embedded, and frozen. Serial (20 μm) sections were cut and stained for c-fos. In brief, sections were incubated for 48 h in a 1:1000 dilution of rabbit c-fos antibody (Cell Signalling Technology, Danvers, MA, USA), with a 1:500 dilution of mouse monoclonal anti-Neu N antibody (Merck Millipore, Darmstadt, Germany) in 10% normal donkey serum. Sections were washed with PBS, then incubated in secondary antibodies of donkey antimouse Alexa Fluor 488 (Thermo Fisher Scientific) and donkey antirabbit Alexa Fluor 546 (1:500; Thermo Fisher Scientific) for 2 h. Sections were washed and mounted with VECTASHIELD Antifade Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA). Images were taken using a ZEISS LSM 710 confocal microscope (Oberkochen, Germany). Cells exhibiting c-fos immunoreactivity were counted in the afferent regions of the spinal cord.

Platonin was synthesized by and obtained from Gwo Chyang Pharmaceuticals (Tainan, Taiwan; Figure 1A). Primary antibody against cleaved-caspase-3 was purchased from Abcam (Cambridge, UK). The anti-Iba1 antibody was purchased from Wako Chemicals USA (Richmond, VA, USA). The anti-NeuN antibody was from Merck Millipore (Darmstadt, Germany). For immunostaining, CF488A donkey anti-mouse IgG and CF488A donkey anti-rabbit IgG were purchased from Biotium (Fremont, CA, USA). Dihydroethidium (DHE) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Platonin was dissolved in phosphate-buffered saline (PBS) and stored at 4 °C.

Six-to-eight-week-old male A_2AR-tag C57BL/6J mice were anesthetized and perfused first with PBS and then with 4% PFA in PBS (pH 7.4). The brains were quickly removed, post-fixed in 4% PFA overnight, and then cryoprotected in 30% sucrose solutions in PBS for 3 days. The brain was cut into 30-μm-thick sections using a microtome (Leica CM1950) and preserved in PBS. Free-floating sections were blocked in blocking solution (0.3% Triton X-100 in PBS and 5% normal donkey serum) for 1 h at room temperature, incubated with the primary antibody in antibody solution (3% normal donkey serum, 0.3% Triton X-100 in PBS) overnight at 4°C, washed with PBS (3 × 10 min), incubated for 2 h at room temperature with the secondary antibodies (1: 500, No. A-11003, Invitrogen) and DAPI (No.C1006, Beyotime, China), and finally washed with PBS (3 × 10 min). The following antibodies were used: anti-HA antibody (1: 200, No.3724, Cell Signaling Technology, United States), anti-NeuN antibody (1: 1,000, No. MAB377, EMD Millipore, United States) and anti-c-Myc antibody (1:50, 1:200 or 1:500, No. 9E10, Santa Cruz Biotechnology, United States). Images were acquired with a Leica DM6B microscope or a Zeiss LSM 880 NLO confocal microscope.