Immunostaining was conducted following established protocols [23 (link)]. Briefly, brain tissues were collected after cardiac perfusion with cold 4% paraformaldehyde (PFA), placed in 4% PFA overnight, and then dehydrated using 30% sucrose/PBS for 4 days. Tissues at distances of − 2.5 to − 5 mm from bregma were selected for serial sections of 25-μm thickness. Sections were incubated overnight with primary antibody, including anti-NeuN (1:200, MAB377, Abcam), anti-cleaved caspase-3 (1:400, #9661, CST), anti-Iba-1 (1:1000, 019–19741, Wako, Japan), anti-Iba-1 (1:500, ab5076, Abcam), anti-CD68 (1:1000, ab125212, Abcam), and anti-CD206 (1:500, AF2535, R&D, USA), and then incubated for 2 h at room temperature with the appropriate fluorescent secondary antibody (Jackson ImmunoResearch Laboratories). DAPI (Southern Biotech) was utilized for nuclear staining and mounting. Micrographs were taken using a confocal microscope (Olympus, Japan). Three sections per rat were used, and three random fields in the ipsilateral basal cortex were acquired in each section, and the immunopositive cells in the basal cortex were quantified using Image J software [24 (link)].
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