Immunofluorescence and Immunohistochemistry Analysis of Neointimal Hyperplasia

For immunofluorescence analysis, the paraffin tissue sections were cut, mounted, deparaffinized, and rehydrated. After blocking, the sections were incubated with primary antibodies against CD45 and Mac2 (Abcam; Cambridge, UK) in 3% BSA overnight at 4 °C, followed by incubation with the corresponding secondary antibody for 1 h at 25 °C, and nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). The CD45- and Mac2-stained cells within the neointima were directly counted in four high-power fields, and the mean numbers of cells were then compared [38 (link)]. For immunohistochemical staining, primary antibodies against MMP-9 (Sigma-Aldrich) and α-SMA (Abcam) were used. After overnight incubation, the tissue sections were washed and incubated using EnVision system-horseradish peroxidase-labeled polymer (Dako; Glostrup, Denmark) for 1 h at room temperature. The sections were visualized with 3,3′-diaminobenzidine tetrahydrochloride (Dako) and counterstained with hematoxylin. The MMP-9-stained cells were counted in four high-power fields and compared. The α-SMA-stained area in neointima was analyzed using ImageJ software and expressed as ratio of positive area as Picro Sirius red stain. For both immuno-staining examinations, three sections in the most severe neointimal hyperplasia region, 200 μm apart, were used for each staining.