Primary monoclonal anti gfap mouse antibody

Cells on 25 mm poly-L lysine-coated glass coverslips were rinsed twice with PBS, pH 7.2-7.4, fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, rinsed three times with PBS, incubated with PBS containing 0.3% Triton X-100 for 30 min, blocked in 10% goat serum in PBST for 1 hr, incubated with primary monoclonal anti-GFAP mouse antibody (Sigma, St. Louis, MO, USA) overnight at 4°C, rinsed three times with PBS, incubated with secondary goat anti-mouse Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) for 2 hr at RT, rinsed three times with PBS, mounted with glycerin, and examined under confocal microscope (Leica, USA) or inverted fluorescence microscope IX51 (Olympus, Japan).

Astrocytic cultures were fixed with 4% formaldehyde in phosphate buffered saline (PBS) and treated with blocking buffer containing 0.3% Triton X-100 and 3% goat serum in PBS. The cells were then incubated with a primary monoclonal anti-GFAP mouse antibody (1:500, Sigma) and stored in a refrigerator overnight at 4°C. After rinsing with PBS, the cells were incubated with a secondary FITC-labeled anti-rabbit IgG antibody (1:500, Sigma) in blocking buffer for 2 h at room temperature. After washing the cells with PBS, fluorescent staining was analyzed under standard FITC settings (488 nm excitation, 585 nm emission) using digital fluorescence microscopy. Cell dishes not exposed to the primary antibody were used as negative controls.