VEGF Immunostaining in Rat Ischemic Brain

Immediately after 2-h ischemia, rat was perfused with ice-cold PBS followed by 4% PFA. Analysis of VEGF was carried out by immunostaining using the 20-μm-thick cryosection as described (Wang et al., 2017 (link)). In brief, tissue was pre-incubated for 1 h at room temperature in PBS containing 0.1% Triton X-100, 1% BSA, and 5% goat serum to block non-specific binding sites. Then anti-VEGF antibody (1:200, Abcam), anti-NeuN (1:200, Millipore), anti-GFAP (1:2,000, Millipore) primary antibody were applied to the sections and incubated at 4°C overnight. Cy3 conjugated secondary antibody (anti-mouse, 1:800) or 488-conjugated secondary antibody (anti-rabbit, 1:800) was incubated with the brain cryosection for 2 h at room temperature. The staining was visualized under LSM 700 confocal laser-scanning microscope (Zeiss), and images were taken from the ischemic and the mirrored non-ischemic region.