2.4. Immunohistochemistry Staining

CD31 immunohistochemistry (IHC) staining was used to identify vasculature in tumor sections. Both 231 WT and 231 VEGF tumors were harvested, fixed in formalin, and embedded with paraffin. Tumor tissue slides were sectioned at 5 µm thickness, then dewaxed and rehydrated. Antigen retrieval was achieved by boiling the slides in pre-warmed citrate buffer, pH 6.0 solution for 20 min. Peroxidase blocking and serum free protein blocking were performed on slides prior to overnight incubation at 4 °C with a rat monoclonal CD31 antibody (platelet endothelial cell adhesion molecule-1, PECAM-1 DIA 310, clone SZ31, Rat IgG2A, Dianova, Hamburg, Germany, 1:30 dilution). Horseradish peroxidase (HPR) conjugated secondary antibody (Vector Laboratories, Burlingame, CA, USA) was used to recognize the primary antibody. After incubation with secondary antibody for 1 h, DAB (3,3′-diaminobenzidine) chromogen was used to develop color, following which slides were counter stained with hematoxylin (Vector Laboratories, Burlingame, CA, USA).

Stained tissue slides were scanned with an Aperio ScanScope XT slide scanner (Aperio Technologies, Vista, CA, USA) and analyzed by Aperio Imagescope. The number of strongly positive pixels normalized to the total number of pixels was obtained. Analyses were performed using the entire histological section.