Anti fluorescence quenching mounting medium

After 24 h of reperfusion, the rats were anesthetized and perfused transcardially with a physiological saline solution followed by 4% paraformaldehyde in PBS. The brains were rapidly removed and post-fixed with 4% paraformaldehyde overnight at 4 °C. Each brain tissue was sliced into 20 μm coronal sections, preincubated in 0.3% Triton X-100 in PBS for 30 min, and then incubated with primary antibodies against LC3B (1:200, Proteintech Group, Wuhan, China) and NeuN (1:1000, Abcam, Cambridge, UK) or p62 (1:200, Proteintech Group) and NeuN overnight at 4 °C. After rinsing 3 times with PBS, the sections were incubated with fluorescent (Cy3)-labeled goat anti-rabbit IgG and fluorescent (FIFC)-labeled goat anti-mouse IgG (1:200, Servicebio) for 2 h at room temperature. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) for 5 min without light. The sections were mounted on a coverslip with an anti-fluorescence quenching mounting medium (Beyotime Biotechnology, Shanghai, China). All sections were observed with a fluorescence microscope (Leica, Wetzlar, Germany) by an investigator who did not know the experimental design. The fluorescence intensity of positive cells in three non-overlapping fields (400×) was calculated using ImageJ software.

Brain tissue sections were processed in the same way as immunohistochemical staining. Sections were mixed with goat polyclonal C3d antibody (cat. no. AF2655, 1:100, RD system, USA), mouse polyclonal GFAP antibody (cat. no. BM0055, 1:200, Boster biological technology, China), and rabbit polyclonal S100a10 antibody (cat. no. 11250–1-AP, 1:100, Proteintech, China), and were incubated overnight at 4 °C. After removing the sections and washing 3 times, the sections were incubated with donkey anti-mouse, donkey anti-rabbit, and donkey anti-goat secondary antibodies labeled with Alexa Fluor 488 and Alexa Fluor 647 (1:200, Jackson, UK) for 1 h at room temperature. After rinsing 3 times, the slides were mounted with an anti-fluorescence quenching mounting medium (Beyotime Biotechnology, China) containing 4’, 6-diamino-2-phenylindole (DAPI), and images were acquired using a fluorescence microscope ((Nikon Eclipse Ti-S, Japan).

Neurobasal‐A medium, B27, GlutaMAX‐L, trypsin (TP), and fetal bovine serum (FBS) were purchased from Gibco Company; Dulbecco's modified Eagle's medium (DMEM) (high glucose, containing sodium pyruvate) was purchased from Biosharp Company; Penicillin–Streptomycin (PS) was purchased from Hyclone Company; Poly‐l‐lysine (PLL) was purchased from Solarbio Company; Neuronal β‐III Tubulin (Tuj1) antibody was purchased from abcam Company; and antifluorescence Quenching mounting medium and 4’,6‐diamidino‐2‐phenylindole (DAPI) were purchased from Beyotime Company. B2 The Biosafety cabinet (Thermo), dissecting microscope, and inverted phase‐contrast microscope were manufactured by Nikon Corporation.