2.9. Immunohistochemical Analysis of NRF2

Four μm thick sections were dewaxed with xylene and hydrated using sequential ethanol washes (100, 95, 85, and 75%), ending with a distilled water wash. Antigen retrieval was performed by heating sections in 0.01 M sodium citrate buffer (pH 6.0). Tissue slides were incubated overnight with NRF2 antibody (dilution 1: 400) at 4°C prior to introduction of secondary antibodies. Sections were incubated with 3,3′diaminobenzidine (DAB) to produce a brown product and counterstained with hematoxylin. The positive staining was evaluated. Five fields of view were randomly selected for each slice under a 400-fold microscope. Image-Pro Plus 6.0 software was used to conduct mean density analysis.