Immunofluorescence Staining

Primary microglial cells and brain tissue sections were identified by immunofluorescence staining. For primary microglial cells, the coverslips were washed with phosphate-buffered saline (PBS) three times and fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, the coverslips were incubated in 0.2% Triton-100 for 10 min and blocked with 5% bovine serum albumin (BSA) for 1 h. For brain tissue sections, 3 days after t-MCAO, mice from each group (n = 3) were deeply anesthetized and transcardially perfused with heparinized saline followed by 4% neutral-buffered paraformaldehyde. Brains were removed and fixed with the same solution for 12 h. Frozen sections of the brain, 5 μm thick. Then, the cells were incubated with anti-IBA-1 mouse monoclonal antibodies (sc-32725, Santa Cruz, CA, USA, 1:200,) and brain sections were incubated with anti-IBA-1 (48934s, Cell Signaling Technology, MA, USA, 1:200) at 4 °C overnight. Coverslips and sections were washed with PBS and incubated with FITC-conjugated goat anti-mouse IgG (A22110 and A22120, respectively, Abbkine, Redlands, CA, USA 1:200) for 2 h. Nuclei were stained with DAPI (C1006, Beyotime, Shanghai, China) for 5 min at room temperature. Images were captured with a fluorescence microscope (Eclipse Ti-S; Nikon, Tokyo, Japan).