Two weeks after surgery, the ischemic limb muscle tissue sections were fixed in 4% paraformaldehyde and permeabilized with xylene. After blocking (5% BSA in PBS), the sections were immunolabeled with primary antibody: CD31 (1 : 100; Abcam) and then incubated with Alexa Fluor ®488 donkey anti-mouse secondary antibody (1 : 250; Invitrogen) after washing. The nuclei were counterstained using DAPI (Invitrogen), and the cells were observed under a fluorescence microscope (Olympus, Japan).

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