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PSA, a classic ARG responsive to both androgen and estrogen, was used to assess the effect of resveratrol on steroid hormone-dependent pathways. Paraffin-embedded sections were deparaffinized into ethanol. Endogenous peroxidase activity was blocked using 0.6% H202 in methanol. Slides were briefly rinsed in PBS prior to application of the serum block (Rabbit Elite ABC Kit, Vector Laboratories, Burlingame, CA, Kit# PK-6101).
Slides were incubated with 250 μg/ml of rabbit polyclonal anti-Human PSA (Dako, Carpinteria, CA, Ref. A0562, Lot 00012666, 3.0 g/l) in 0.1% bovine serum albumin–PBS overnight at 4°C and rinsed in PBS; secondary antibody (Rabbit Elite Kit) was then applied as directed. Slides were again rinsed in PBS, ABC reagent (Rabbit Elite Kit) was applied, a final PBS wash was performed and slides were stained with 3,3-diaminobenzidine tetrahydrochloride (Sigma–Aldrich, St. Louis, MO, 10 mg of substrate/tablet). Slides were counterstained with hematoxylin. Human prostate tissue was used as the positive control.
Proliferating cell nuclear antigen (PCNA) was used as a proliferation marker. Freshly cut paraffin-embedded sections were deparaffinized into ethanol. Endogenous peroxidase activity was blocked using 0.6% H2O2 in methanol. Antigen retrieval was performed by microwaving in deionized water for two 5 min cycles. Slides were cooled for 20 min and rinsed in deionized water. Monoclonal mouse anti-PCNA (Dako, clone PC10, code number M0879, 570 mg/l) was diluted to 0.7 μg/ml. Following a final PBS wash, slides were stained with 3,3-diaminobenzidine tetrahydrochloride (Sigma–Aldrich, 10 mg of substrate/tablet). Slides were counterstained with hematoxylin. Mouse small intestine tissue was used as a positive control.
Paraffin-embedded sections were deparaffinized into PBS. An apoptosis detection kit using the terminal deoxynucleotidyl transferase dUTP nick and labeling assay was used (ApopTag Peroxidase In Situ Apoptosis Detection Kit, Chemicon International, Temecula, CA) according to the manufacturer’s instructions. Mouse testes tissue was used as a positive control.
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