NeuN/TUNEL staining and immunofluorescence analysis
The rats were killed through chloral hydrate administration and cardiac perfusion with physiological saline and 4% paraformaldehyde. Brains were harvested and fixed in 4% paraformaldehyde for 48 h, followed by dehydration in 30% sucrose. A cryostat vibratome was used to obtain 20-μm-thick brain coronal sections. The brain sections were blocked with 0.3% (w/v) BSA in PBS at room temperature for 1 h. Sections were incubated using NeuN antibodies (Millipore, 1:1000) overnight at 4°C. Slides were washed in TBST, incubated with Alexa Fluor 488 AffiniPure Donkey antibody, and counterstained with DAPI. The number of NeuN/TUNEL-positive cells in the ischemic region was calculated using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA).
The brain coronal sections were incubated using primary antibodies against CD11b (NB100-65284) and C1QTNF6 (Abcam, ab36900) at 4°C overnight, followed by incubation with a fluorophore-labelled secondary antibody (Santa Cruz Biotechnology). Fluorescence images were acquired using an Olympus Fluoview FV1000 fluorescence microscope (Olympus, Japan).