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Paraffin-embedded sections were deparaffinized into ethanol. Endogenous peroxidase activity was blocked using 0.6% H2O2 in methanol. Antigen retrieval was performed by microwaving for two 10 min cycles in citrate buffer. Slides were cooled for 20 min at room temperature, followed by a 20 min serum block (Goat Elite ABC Kit, Vector Laboratories, Kit# PK-6105). Anti-mouse vascular endothelial growth factor (VEGF) antibody, P20 from Santa Cruz Biotechnologies (sc1836, lot A1405, goat polyclonal IgG, 200 μg/ml), was used. Slides were incubated with 0.2 μg/ml of P20 in 0.1% bovine serum albumin–PBS for 60 min at room temperature.
Slides were rinsed in PBS (0.05% Tween); secondary antibody (Goat Elite Kit) was then applied as directed. Slides were again rinsed in PBS (0.05% Tween), ABC reagent (Goat Elite Kit) was applied, a final PBS wash was performed and slides were stained with 3,3-diaminobenzidine tetrahydrochloride (Sigma–Aldrich, 10 mg of substrate/tablet). Slides were counterstained with hematoxylin. Mouse kidney tissue was used as a positive control. Five samples from each diet group were evaluated by a certified pathologist to obtain qualitative measurement of VEGF expression.
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Chuck Reynolds
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