Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining

The 4-µm-thick sections of cerebral tissue were deparaffinized and rehydrated, and subsequently analyzed using terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining to label the nuclei of apoptotic cells. Sections were first incubated in 3% H₂O₂ and then in the TUNEL reaction mixture (Roche Applied Science, Penzberg, Germany), according to the manufacturer’s instructions. The sections were rinsed and visualized using 3,3’-diaminobenzidine (DAB). Hematoxylin was used for counter-staining. The positive cells were observed under a light microscope (Olympus, Tokyo, Japan). The apoptosis index was calculated as the percentage of TUNEL-positive cells relative to the total number of cells.