Immunofluorescence Labeling of Brain Cells

Immunofluorescence was performed as previously described (Jing et al., 2015 (link)). Briefly, sections were processed for immunofluorescence labeling with CD34 antibody (for endothelial cells of microvessels), glial fibrillary acidic protein (GFAP) antibody (for astrocytes), NeuN antibody (for neurons) and cleaved caspase-3 (C-caspase 3) antibody (for apoptotic cells). First, sections were immersed in blocking solution (5% normal goat serum or donkey serum in PBS) at room temperature for 2 h, then incubated overnight at 4°C with CD34 antibody (Rabbit, 1:200, Boster, Wu Han, China), GFAP antibody (Rabbit, 1:1000, Abcam, United Kingdom) separately, and NeuN antibody and cleaved caspase-3 antibody (goat or mouse, 1:1000, Abcam, Cambridge, MA, USA) for double-labeling. After three washes with 0.01 M PBS, the sections were then incubated with secondary antibody (Alexa Fluor 488-conjugated goat anti-rabbit, donkey anti-rabbit, or donkey anti-mouse IgG and Alexa Fluor 546-conjugated donkey anti-goat, Jackson Immunoresearch, West Grove, PA, USA) for 2 h at room temperature. The concentration of all secondary antibodies was 1:1000. After three washes in PBS, sections were covered with anti-quenching fluorescence mounting medium.