Immunohistochemistry of Endothelial and Mesenchymal Markers

Tissue preparation and immunohistochemistry were performed as previously described (33 (link)). The primary antibodies used were: anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) monoclonal antibody (1:100; BD Pharmingen, San Diego, CA, USA), anti-SMA antibody (1:200; Sigma-Aldrich), anti-EphrinB2 polyclonal antibody (1:1,000), and anti-Snail polyclonal antibody (1:2,000) (both from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). Secondary antibodies were biotinylated rabbit anti-rat (1:500) and goat anti-rabbit (1:200) antibodies from Vector Laboratories, Inc., Burlingame, CA, USA. The peroxidase activities were visualized using streptavidin-horseradish peroxidase (HP) and the diaminobenzidine (DAB) detection system (Vector Laboratories, Inc.). The slides were then counterstained with hematoxylin (Surgipath; Leica Microsystems, Wetzlar, Germany).