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Differential effects of Resveratrol on androgen-responsive LNCaP human prostate cancer cells in vitro and in vivo. P-9

IHC staining protocol for platelet/endothelial cell adhesion molecule-1

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.

Article Produced By

Diet, Genomics and Immunology Laboratory, Beltsville Human Nutrition Research Center, Agriculture Research Service, United States Department of Agriculture, 10300 Baltimore Avenue, Building 307C, Room 132, Beltsville, MD. Laboratory of Cellular Regulation and Carcinogenesis, National Cancer Institutes, National Institutes of Health, Department of Nutrition and Food Science, University of Maryland, College Park, Department of Pharmaceutical Sciences and Pharmacology and Toxicology Graduate Program College of Pharmacy, Washington State University, Pullman, National Food Research Institute, Tsukuba, Ibaraki 305-8642, Japan. Nutritional Sciences Research Group, Division of Cancer Prevention, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Biometrical Consulting Service, Beltsville Area, Agriculture Research Service, United States Department of Agriculture, Beltsville, MD 20705, USA. Division of Nutritional Sciences, University of Texas at Austin, Austin, TX 78712, USA. Department of Carcinogenesis, MD Anderson Cancer Center, Smithville, TX 78957, USA
 

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