Immunofluorescent staining

Immunofluorescent labeling was performed as previous studies to detect GFAP and IBA1 in hippocampal CA1 region (Lai et al., 2021). Briefly, mice were sacrificed under deep anesthesia and transcardially perfused with PBS and 4% paraformaldehyde (PFA) (SJ-BL539A, Biosharp, China). Brains were harvested and fixed in 4% PFA at 4°C for 24 h, dehydrated, and embedded in optimal cutting temperature compound. Coronal 20-μm thick sections were cut and mounted on microscope slides. After being washed in PBS, sections were blocked in 5% goat serum (AR0009, Boster, China) in PBS containing 0.3% Triton-X 100 (ST795, Beyotime, China) for 1 h at room temperature (RT) and then incubated at 4°C overnight with primary antibodies: mouse monoclonal anti-GFAP antibody (1:50, Beyotime, AF0156, China) and rabbit monoclonal anti-IBA1 antibody (1:100, ab178847, United States). Sections were rinsed in PBS with 0.3% Triton-X 100. The goat anti-mouse IgG antibody conjugated fluorescein isothiocyanate (1:500, Beyotime, A0568, China) and goat anti-rabbit IgG antibody conjugated cyanine 3 (1:500, Beyotime, A0516, China) were incubated with sections for 2 h at RT in the dark. The sections were washed in PBS, incubated with 4’,6-diamidino-2’-phenylindole (Servicebio, G1012, China) for nuclear staining, rinsed and mounted with antifade mounting medium (Beyotime, P0128M, China). Images of immunostaining were acquired by LSM 800 microscope system (LSM 800 with aiyscan, Zeiss, Germany), and a negative control omitting the incubation with the primary antibody was included in all experiments. The positive stained area for one interested marker was quantified using the Image J (National Institutes of Health, Bethesda, MD, United States), and presented as ratio of average fluorescence intensity and fluorescence area. All quantitative analyses were performed in a blinded manner.