2.5 TUNEL staining
Terminal Deoxynucleotidyl Transferase (TdT)-mediated dUTP Nick-End Labeling (TUNEL) (Beyotime, Shanghai, China) staining was used to sight neuronal apoptosis40. In brief, cells and brain sections were fixed with freshly prepared 4% paraformaldehyde for 20 minutes at room temperature and washed twice with PBS. Subsequently, to inhibit endogenous peroxidase activity in the brain, the sections were incubated with a methanol solution containing 0.2% H2O2 for 30 minutes. Subsequently, the samples were treated with 50 μl of TUNEL reaction mixture and incubated in a dark and humidified atmosphere at 37 ℃ for 60 minutes. The nuclei were then stained with 4′,6-diamidino-2-phenylindole (DAPI). The images were acquired using a fluorescence microscope (Olympus, Tokyo, Japan). The results were expressed as the apoptosis index, which was calculated by dividing the number of TUNEL-positive cells by the total number of cells in five randomly selected fields for each condition and multiplying by 100%.