Immunofluorescence Staining of Brain Sections

Immunofluorescence staining was performed as described previously [30 (link), 31 (link)]. Briefly, the brain sections were prepared as above. The rehydrated sections were incubated in Triton X-100 for 30 min, and were blocked with 1% bovine serum albumin (BSA) for 1 h at 37°C. The sections were then incubated overnight at 4°C with the following primary antibodies: rabbit anti-mouse CCL20 antibody (Abcam Biotechnology, Cambridge, UK), mouse anti-mouse Iba1 antibody (Santa Cruz Biotechnology), mouse anti-mouse NeuN antibody (Abcam Biotechnology), and mouse anti-mouse GFAP antibody (Abcam Biotechnology). After washing with TBST, the sections were incubated with goat anti-rabbit/mouse secondary antibody (Thermo Fisher Scientific) at 37°C for 1 h. Cellular nuclei were stained with DAPI (Beyotime, Jiangsu, China) at 37°C for 10 min. Images were captured using a microscope with a digital camera (Olympus, Tokyo, Japan).