Apoptosis assay

The apoptosis was assayed by using Tunel apoptotic cells detection kit in strict guidance with the in situ apoptosis detection kit instructions. Anti-vwF (endothelial cell markers) antibody (1:200) was added to the slide at 4°C overnight in a wet box. Then the corresponding peroxidase-labeled secondary antibody (1:250) was added at 4°C for 1 h in dark room. DAB was used for coloration. Eight high visions (×200) were randomly selected from each section on ischemia and total number of cells and apoptosis cells were counted. Apoptotic index (AI) was calculated according to a percentage of apoptosis cell in the total endothelial cells.