Ix51 reflected light fluorescence microscope

Terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining was assessed by the In Situ Cell Death Detection Kit, POD (Hoffmann-La Roche Ltd., Basel, Switzerland). After 24-hour reperfusion, the sections were prepared, and the staining was performed according to the protocol provided by the manufacturer. All sections were counterstained with 4′,6-diamidino-2-phenylindole (Thermo Fisher Scientific). In each case, ten fields in the infarcted cortex were randomly selected for apoptotic cell counting in a blinded manner using an Olympus IX51-reflected light fluorescence microscope (Olympus Corporation, Tokyo, Japan). The extent of apoptosis was calculated and expressed as a ratio of TUNEL-positive neurons versus total neurons.

Neuronal apoptosis was assessed by TUNEL staining using a commercial in situ cell death detection kit (Nanjing KeyGEN Biotech Co. Ltd, Nanjing, People’s Republic of China) according to the manufacturer’s instructions. All sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen, Carlsbad, CA, USA). In each case, ten fields in the infarcted cortex were randomly selected for apoptotic cell counting in a blinded manner using an Olympus IX51 reflected light fluorescence microscope (Olympus, Japan), and the percentage of TUNEL positive cells (TUNEL/DAPI) was calculated.