4.7. Immunohistochemistry (IHC) and Immunofluorescence Analysis

For IHC staining, the paraffin slices were incubated with primary antibodies to NF-κB (1:200), TNF-α (1:200), iNOS (1:200), and COX-2 (1:200), respectively, overnight at 4 °C in a humidified chamber, followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at 37 °C. A brownish yellow color in the cytoplasm or nucleus was regarded as positive cellular staining. The image was photographed using a light microscope and the intensity of cells’ positive expression was quantified by Image-Pro Plus 6.0. For immunofluorescence staining, the kidney slices were incubated with primary antibodies to CYP2E1 (1:200) and HO-1 (1:200), respectively, overnight at 4 °C in a humidified chamber, followed by incubation with DyLight 488-labeled secondary antibodies for 1 h at 37 °C. DAPI (4,6-diamidino-2-phenylindole) was used for nuclear staining. The image was photographed with a fluorescence microscope (Leica TCS SP8, Solms, Germany) and the intensity of the immunofluorescence was quantified by Image-Pro Plus 6.0.