Immunohistochemical Staining of PECAM-1 and vWF

Immunohistochemistry was performed by employing the avidin–biotin complex (ABC) method as has been described previously^{6 (link), 61} . Briefly, FFPE sections were deparaffinized, rehydrated and then microwaved for 30 min in citrate buffer (pH 6.0). Sections were incubated for 5 min with 3%H₂O₂ in methanol and then blocked for 1 h at RT with PBS containing 5% normal goat serum and 0.1% Triton X-100. Thereafter, they were incubated for 2 h at RT with primary antibodies. The primary antibodies used in this study included the rabbit anti-platelet endothelial cell adhesion molecules-1 (PECAM-1; 1:400) and rabbit anti-von Willebrand factor (vWF; 1:400, all obtained from Abcam, Cambridge, UK). After being washed, sections were incubated for 45 min at RT with biotinylated secondary antibody (1:200; Vector Laboratories, CA, USA), followed by peroxidise coupled ABC (Thermo Fischer Scientific, Waltham, MA, USA). Antibody binding was visualized using DAB-H₂O₂ for 5 min at RT followed by being counterstained with hematoxylin. They were then dehydrated, mounted with coverslips and observed under a light microscope. Scoring of immunolabeling positive cells for each antibody in different anatomical regions was done according to the method previously described^{6 (link)}.