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Micro blood vessels were identified using platelet/endothelial cell adhesion molecule-1 (PECAM-1) staining (33). Paraffin-embedded sections were deparaffinized and placed in water. Slides were postfixed in zinc fixative (BD Pharmigen, San Jose, CA) for 10 min followed by a deionized water and PBS rinse. Endogenous peroxidase activity was blocked using 0.6% H2O2 in methanol.
Antigen retrieval was performed by microwaving the slide for two 10 min cycles in 1 mM ethylenediaminetetraacetic acid buffer. Slides were cooled for 20 min at room temperature, followed by a 30 min avidin–biotin block (Vector Avidin–Biotin Blocking Kit). Slides were briefly rinsed in PBS prior to application of the serum block (Goat Elite ABC Kit, Vector Laboratories, Kit# PK-6105).
Slides were incubated with 1 μg/ml of anti-PECAM-1 antibody (M-20, sc-1506, goat polyclonal IgG, 200 μg/ml, Santa Cruz Biotechnologies, Santa Cruz, CA) in 0.1% bovine serum albumin–PBS for 30 min at room temperature and rinsed in PBS; secondary antibody (Goat Elite Kit) was then applied as directed. Slides were rinsed in PBS, ABC reagent (Goat Elite Kit) was applied, a final PBS wash was performed and Vector Nova Red (Vector Nova Red Substrate Kit, for Peroxidase, sk-4800) was applied. Slides were counterstained with hematoxylin. Mouse kidney and heart tissues were used as positive controls.
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