Apoptotic cells were examined with TUNEL assay (Yeasen) according to the manufacturer’s instructions. In brief, brain slices were digested for 10 min by proteinase K (20 μg/ml) at room temperature after IF staining. After washing twice with PBS, brain slices were incubated with equilibration buffer for 30 min at room temperature, and subsequently with Alexa Fluor 488-12-dUTP Labeling Mix for 60 min at 37 °C. After washing with PBS for three times, brain slices were stained with DAPI (2 μg/ml, Roche, Basel, Switzerland) before being mounted under coverslips.