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%.