Image pro plus software version 6

Aortic and kidney nitrotyrosine as well as aortic F4/80 localization and expression were determined by immunohistochemistry. In brief, 4-µm paraffin sections mounted on Superfrost slides were dewaxed, and endogenous peroxidases were inactivated with 3% H₂O₂ in Tris-buffered saline. Antigen retrieval was performed for kidney sections. Thereafter, sections were incubated with a serum blocking agent and a biotin-avidin blocking kit (Vector Laboratories). Primary antibodies were added to sections and incubated overnight at 4°C. The next day, secondary antibody, biotinylated anti-rabbit Ig, or the biotinylated anti-rat secondary antibody was added for 30 min followed by horseradish peroxidase–conjugated streptavidin (1:500), incubated for 3 min in 3,3′-diaminobenzidine tetrahydrochloride, and counterstained with hematoxylin. All sections were examined under an Olympus BX-50 light microscope (Olympus Optical) and digital quantitation (Image-Pro Plus software version 6.0) and assessments were performed in a blinded manner. Nitrotyrosine staining was expressed as postively stained area over total area of the section while F4/80 positive cells were counted and averaged over sections.

Renal injury was assessed by measuring urinary albumin at midpoint (10 weeks of diabetes) and endpoint (20-weeks of diabetes) and PAS staining to quantify mesangial expansion. Urinary albumin was measured using a mouse albumin ELISA kit (Bethyl Laboratories) as per the manufacturer's instructions. PAS staining was performed and counterstained with hematoxylin. All sections were examined under an Olympus BX-50 light microscope (Olympus Optical) with digital quantitation (Image-Pro Plus software version 6.0) and assessments performed in a blinded manner.