Immunohistochemical Analysis of Oxidative Stress

Kidney tissues were fixed in formalin and 70% ethanol, embedded in paraffin and cut into 5-µm sections. Immunostaining was performed using the avidin biotin complex (ABC) method with 0.1% diaminobenzene solution used as the chromogen as previously described^{52 (link),53 (link)}. Antigen retrieval treatment with sodium citrate buffer (pH 6.0) was applied at 90–95 °C for 30 min. Non-specific binding was blocked in a buffer containing 10% normal goat serum, 1% Triton-X in PBS for 30 min. Kidney sections were incubated with the rabbit polyclonal 4-hydroxy-2-nonenal Michael Adducts (4-HNE; dilution: 1:10,000; Calbiochem, La Jolla, CA), rabbit polyclonal NOX-4 antibody (dilution: 1:200; Proteintech, Rosemont, IL, USA) and secondary biotinylated goat anti-rabbit antibody (dilution: 1:400; Vector Laboratories, Burlingame, CA, USA). Slides were scanned with a Hamamatsu Nanozoomer 2.0 HT utilizing NDP.scan and NDP.view as the imaging software at 0.25 and 20X magnification. Images of 4-HNE or NOX-4 staining were analyzed using Adobe Photoshop 7.0 in 9 sections from each kidney cortex (n = 4–5) by an investigator blinded to the experimental groups and the data are reported as relative intensity units.