2.10. TUNEL Staining Assay

To measure the extent of neuronal apoptosis in cerebral I/R injury, an in situ apoptosis detection kit (POD, Roche, and Mannheim, Germany) was employed to discover neuronal apoptosis caused by ischemia. A TUNEL analysis was carried out with minor modification according to the manufacturer’s instructions. Firstly, the sections were installed on the slides and permeabilized by incubating them with 100 µL of 20 µg/mL proteinase K solution for 15 min. Next, the sections were incubated with 100 µL of 0.3% H₂O₂ for 5 min and incubated by equilibration buffer and terminal deoxynucleotidyl transferase to inactivate endogenous peroxidases. Then, anti-digoxigenin-peroxidase conjugates were employed to incubate the sections. Finally, the utilization of diaminobenzidine demonstrated peroxidase activity in all tissue sections, and the slices were counterstained with hematoxylin. TUNEL-positive cells were visualized by using a Leica microscope (Leica DM4000B, Germany), and images were randomly selected for image analysis via ImageJ software.