Tunel regent

The number of apoptotic cells was assessed using an apoptosis detection kit (Roche Diagnostics) in accordance with the manufacturer's protocols. Briefly, the cells were fixed in 4% paraformaldehyde at 37˚C for 15 min and incubated with proteinase K (Beyotime Institute of Biotechnology) at room temperature for 15 min. Subsequently, the cells were placed in 3% H₂O₂ at room temperature for 15 min, cultivated with TUNEL regent (Beyotime Institute of Biotechnology) at 37˚C for 1 h and counterstained with DAPI for 5 min at room temperature. The number of positive cells was mounted with fluorescent mounting media (Beijing Solarbio Science & Technology Co., Ltd.) and analyzed using ImageJ 1.8.0 software (National Institutes of Health). Overall, >10 fields of view/section for each sample were assessed. A fluorescence microscope was used for the visualization of the positive cells.

Paraffin sections of brain tissues were dewaxed and dehydrated with gradient ethanol and washed with PBS (Thermo Fisher Scientific, Shanghai, China) for 5 min×3 times. Then, the sections were incubated with proteinase K at 37℃ for 30 min, followed by washing with PBS for 5 min×3 times. Subsequently, the TUNEL regent (Beyotime) was added and incubated for 30 min at 37℃. After washing with PBS, the sections were developed with peroxidase solution and DAB, and then counterstained by hematoxylin. The TUNEL-positive cells were observed under a fluorescence microscope.