2.8.2. Immunofluorescence Staining and Analysis for GFAP and IBA-1

The rest sections were used for immunofluorescence staining for glial fibrillary acidic protein (GFAP, reactive astrocytes), ionized calcium-binding adapter molecule 1 (IBA-1, microglia), and 4′, 6-diamidino-2-phenylindole (DAPI, nuclei). After being blocked by 5% normal goat serum, 1% bovine serum albumin, and 0.5% TritonX in PBS, sections were incubated with rabbit anti-mouse GFAP (1 : 500; Servicebio) or rabbit anti-mouse IBA-1 antibody (1 : 2000; Servicebio) overnight at 4°C. The following day, sections were incubated with fluorochrome-conjugated secondary antibody (goat anti-rabbit, 1 : 200; Servicebio) for 1 hour and DAPI (0.01 mol/L in PBS, Servicebio) for 10 min successively at room temperature and then washed in PBS and mounted. Fluorescent images were captured on a NIKON Eclipse ci microscope. 5-6 representative images of each mouse were taken at 400x magnification in the hippocampus and analyzed by blinded investigators with ImageJ.