Immunofluorescent Staining

Three mice in each group were selected randomly and transcardially perfused with 0.01 M phosphate-buffered saline (PBS, pH 7.4) followed by 4% paraformaldehyde (PFA) in PBS. The whole brains were dissected out and post-fixed in 4% PFA overnight, followed by cryoprotection in 30% sucrose in PBS overnight. After embedded with optimum cutting temperature compound (OCT), the brains were cut into sagittal sections (25 m thick), and then stored at −80°C for immunostaining (Pruski et al., 2019).

Immunohistochemistry was performed as described previously (Zhu et al., 2014) with some modifications. Sections were re-hydrated washed in PBS for 3 × 10 min in a microwave followed by incubation with a blocking solution containing 5 % bovine serum albumin (BSA) and 0.1% Tween-20 in PBS (pH 7.4) for 6 min at 95°C to retrieve the antigen. Then, brain sections were incubated with rabbit anti-Iba-1 (1:1,000; LAK4357, Wako, Japan), rat anti-CD68 (1:200; MCA1957GA, Bio-Rad Company, United States), guinea pig anti-DCX (1:300; AB2253, Millipore, USA), or rabbit anti-GFAP (1:1,000; z0334, Dako, USA), at 4°C overnight. After several washes in PBS, sections were incubated with donkey anti-rabbit Alexa Fluor 488-conjugated IgG (1:500; {"type":"entrez-nucleotide","attrs":{"text":"R37118","term_id":"794574","term_text":"R37118"}}R37118, Invitrogen, Carlsbad, CA, USA), and biotinylated horse anti-rat or horse anti-guinea pig IgG (1:500, Jackson ImmunoResearch, USA) at room temperature for 2 h. Ultimately, brain sections were incubated with streptavidin-Cy3 (1:1,000, 016160084, Jackson ImmunoResearch, West Grove, PA, USA) and counterstained with Hoechst 33258 (1:2,000, 94403, Sigma, St.Louis, MO, USA) at room temperature for 1 h. Fluorescent images were taken on a laser-scanning confocal fluorescent microscope (Leica TCS SP8, Leica Microsystems, Wetzlar, Germany).