Immunohistochemical Analysis of Brain Sections

Brain slice preparation and imaging were completed according to the previously reported methods [22 (link),24 (link)]. The antibodies used were rabbit anti-NeuN (neuronal nuclei antibody; 1:800; Abcam, Cambridge, MA, USA), goat anti-GFAP (glial fibrillary acidic protein antibody; 1:800; Abcam, Cambridge, MA, USA), Cy3-conjugated goat anti-rabbit immunoglobulin G (IgG) (1:400; The Jackson Laboratory, Bar Harbor, ME, USA) and rabbit anti-goat IgG conjugated with Cy3 (1:400; The Jackson Laboratory, Bar Harbor, ME, USA). The sections were incubated with the primary antibody overnight at 4 °C. After washing 3 times with PBS, the slices were incubated with the secondary antibody for 1 h at 37 °C. After washing with PBS, all the brain slices attached to the microscope slides were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (1:4000; Beyotime, Shanghai, China) and sealed with 70% glycerol. Imaging was performed using an Olympus VS120 virtual microscopy slide scanning system (Olympus, Tokyo, Japan) or a Leica TCS SP8 confocal microscope (Leica, Wetzlar, Germany).