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Two additional washes, one in a 2 lysis buffer (1 M NaCl, 2 mM EDTA, 20 mM Tris pH 7.5 supplemented with 0.04% ( v/v ) SDS and 0.05% ( v/v ) Tween-20) and another in 2 lysis buffer supplemented with 20% ( v/v ) RNAse inhibitor, were performed to buffer match the gels and the spacer

Then, the sections were coated with an anti-CD31 antibody (Santa Cruz Biotechnology, Dallas, TX) at 4 C overnight, followed by incubation with an HRP-labeled secondary antibody (Gene Tech, Shanghai, China), and the sections were visualized with a DAB system (Gene Tech)

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