Immunohistochemical Analysis of Arterial Remodeling

Immunohistochemical staining was conducted as previously described [5 (link)]. After fixed with 4% paraformaldehyde, the arteries were perfused with saline and embedded in paraffin. The artery sections (4–5 μm) were obtained at 80-μm intervals and then stained with hematoxylin and eosin (H&E; Solarbio). Finally, the areas of intima and media were measured by using Image-Pro Plus software. For immunohistochemical staining, artery sections were incubated with anti-Ki-67 primary antibody (cat 9449 T; Cell Signaling Technology) after being blocked (4°C, overnight). The sections were then incubated with a secondary antibody (Proteintech, Rosemont, USA) and counterstained with Mayer hematoxylin. Five artery sections per sample were collected for staining.