Gfap monoclonal antibody

The spinal cord was embedded in OCT compound, and frozen sections of the spinal cord were cut horizontally at 10 μm thick, and attached to glass slides. Sections were washed with PBS, and immuno-blocked with 3% BSA in PBS for 30 min at room temperature. For the detection of intracellular proteins, the sections were permeated with 0.3% Triton X-100 in 1% BSA-PBS for 30 min prior to incubation with primary antibodies. The sections were then incubated overnight with a solution containing the primary antibodies as follows: mouse anti-β-III-tubulin monoclonal antibody (1:1,000; Covance) for axons, mouse anti-phosphorylated growth-associated protein 43 (pGAP-43, S96) monoclonal antibody (1:1,000; Wako) for regenerating axons, and mouse anti-glial fibrillary acidic protein (GFAP) monoclonal antibody (1:500; Sigma) or rabbit anti-GFAP polyclonal (1:2,000; Novus Biologicals) antibodies for astrocytes. After being washed with PBS, the sections were incubated overnight with CF488A-labeled goat anti-mouse IgG, CF488A-labeled anti-rabbit IgG, CF555-labeled goat anti-mouse IgG (1:200; Biotium), or Alexa Fluor 555 goat anti-rabbit IgG (1:200; Thermo Fisher Scientific) antibodies.

The effects of the drugs examined on differentiation of NSCs were examined by immunocytochemistry at 72 hours after drug exposure. NSCs were washed with PBS and fixed with 4% paraformaldehyde for 15 minutes. After washing with PBS, NSCs were blocked with PBS and 10% goat serum for 30 minutes at room temperature. NSCs were then incubated in PBS supplemented with 0.4% Triton-X-100 (Sigma) and 0.1% bovine serum albumin. NSCs were re-washed and treated with mouse anti-rat β-tubulin monoclonal primary antibody (1:400; Millipore, Billerica, MA, USA) and rabbit anti-rat glial fibrillary acidic protein (GFAP) monoclonal antibody (Sigma) overnight at 4°C. After washing with PBS, NSCs were incubated with FITC goat anti-rabbit IgG (1:100; Thermo Fisher Scientific Inc., Rockford, IL, USA) and TRITC goat anti-mouse IgG (1:100; Thermo Fisher Scientific Inc.) for 2 hours at room temperature in a wet box. Next, NSCs were incubated with 4′,6-diamidino-2-phenylindole (DAPI) staining solution. Fluorescence signal was observed using a confocal microscope (Carl Zeiss GmbH, Jena, Germany) and the percentage of differentiated cells was determined by flow cytometry (Beckman-Coulter), in accordance with our previously described protocol (Lu et al., 2012a, b; Li et al., 2014).