Immunohistochemical Dual Staining Protocol

The IHC staining was done as previously described [24 (link)]. Mouse on mouse blocking reagent for vimentin staining, ABC-HRP kit and ABC-AP kit were purchased from Vector Laboratories (Burlingame, CA). CD31 antibody (Thermo Fisher Scientific, Reinach, Switzerland) and biotinylated goat anti-rabbit secondary antibody (Vector Laboratories) were diluted 1:50 and 1:200, respectively. Vimentin antibody and biotinylated goat anti-mouse secondary antibody (Vector Laboratories) were diluted 1:200 and 1:800, respectively. CYR61 antibody (Santa Cruz Biotechnology, Inc., Heidelberg, Germany, #sc13100) and biotinylated goat anti-rabbit secondary antibody were diluted 1:150 and 1:600, respectively. For CD31/vimentin double-staining, tissues were first stained with anti-CD31 and biotinylated goat anti-rabbit secondary antibody, followed by anti-vimentin-staining and biotinylated goat anti-mouse secondary antibody. Substrates used were DAB (Sigma-Aldrich) and Vector Red (Vector Laboratory).