Immunohistochemical staining with von Willebrand factor (vWF) was used to detect newly formed capillaries. For this, 6-μm sections were dewaxed, rehydrated, and incubated with proteinase K (S3020; Dako Japan, Tokyo, Japan) for 5 minutes at room temperature for antigen retrieval. Anti-vWF rabbit polyclonal antibodies (1:5000, Code No. A0082; Dako) were used as the primary antibody followed by the secondary antibody (K4003, EnVision; Dako). The staining was visualized using 3-3’-diaminobenzidine-4HCl (DAB, Code 725191; Nichirei Biosciences Inc., Tokyo, Japan); the sections were then counterstained with hematoxylin and micrographs were taken under an optical microscope.
In each section, a rectangle of 500 × 300 μm was selected at the center of the granulation area beyond the muscle layer, and the number of newly formed capillaries in the rectangle and the cross-sectioned area of neovascularization were measured using the BZ-II Analyzer imaging software program (version 1.42; KEYENCE Co.).
In each section, a rectangle of 500 × 300 μm was selected at the center of the granulation area beyond the muscle layer, and the number of newly formed capillaries in the rectangle and the cross-sectioned area of neovascularization were measured using the BZ-II Analyzer imaging software program (version 1.42; KEYENCE Co.).