Terminal deoxynucleotidyl transferase-mediated dUTP nick 3'-end labeling (TUNEL) staining and immunofluorescence staining of CD31

TUNEL staining was performed to detect dying cells using an In Situ Cell Detection Kit (Roche, Nutley, NJ, USA) according to the manufacturer's instructions. TUNEL and immunofluorescence staining of CD31 were combined to assess endothelial cell apoptosis. Briefly, paraffin sections were incubated with anti-CD31 antibody (1:500, Abcam, Cambridge, MA, USA) at 4 °C for 24 h. After 1 h incubation with Cy3-conjugated anti-mouse IgG (1:100, Thermo Fisher Scientific, Waltham, MA, USA), 50 µl of TUNEL mixture was added. Then the sections were continued to incubate at 37 °C for 1 h. At the end, 4',6-diamidino2-phenylindole (Dapi, 1:50000, Kaiji Biological, Nanjing, China) was added and incubated for 4 min. A fluorescence microscope was used to determine the number of apoptotic endothelial cells around the contusion area.

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