D3s3i

For immunostaining, euthanized mice were perfused intracardially with 30 ml pre-cold PBS and 20 ml 4% paraformaldehyde (PFA) sequentially. After post-fixation, the brains were cut coronally at 50 μm thickness by a vibratome (Leica, Buffalo Grove, IL, United States). Sections were permeabilized in PBST (0.1% Triton X-100 in PBS) for 10 min, blocked for one hour at RT in blocking buffer (10% BSA in PBS), and incubated overnight at 4^°C with the following primary antibodies: a mouse monoclonal anti-acetyllysine antibody (1:500, PTM101, PTM Biolab, Hangzhou, China), a rabbit monoclonal anti-NeuN antibody (1:250, D3S3I, Cell Signaling Technology), a rabbit polyclonal anti-Iba-1 antibody (1:1000, 019–19,741, Wako Chemicals GmbH, Neuss, Germany), a rabbit monoclonal anti-GFAP antibody (1:250, Cell Signaling Technology), and a rabbit polyclonal anti-CD31 antibody (1:100, 557355, BD Biosciences, United States). The sections were then incubated with the following secondary antibodies for 2 h at RT: Alexa Fluor 488-conjugated goat anti-mouse IgG and Alexa Fluor 555-conjugated 82071553 (1:200, Cell Signaling Technology). Nuclei were counterstained with DAPI (1 μg/ml, Sigma). Images were captured on a Zeiss LSM710 confocal microscope (Munich, Germany).