Intracellular Nrf2 Localization by Immunofluorescence

The intracellular distribution of Nrf2 was displayed by immunofluorescence technique. Paraffin-embedded tissue sections (4 µm) were dewaxed in xylene and rehydrated in graded ethanol solutions. The antigen retrieval method with citric acid solution was proceeded for 5 min at 95°C, and then was washed in PBS. Follwing that, the tissues were permeabilized with 0.5%Triton X-100 for 15 min and blocked in normal goat serum at room temperature for 30 min [28] (link). After incubation with the polyclonal Nrf2 primary antibody conjugated to FITC (1∶150, Biorbyt, UK) at 4°C overnight, the sections was washed triple with PBS. At last, the nuclei were counterstained with DAPI (Roche, Shanghai, China) and visualized using a fluorescent microscope (Olympus U-25ND25, Tokyo, Japan). The green staining was showed in cytoplasm and nucleus, while blue staining was in nucleus. The overlay color was considered to be positive. Ultimately, the results were evaluated by semi-quantitative analysis based on the proportion of the Nrf2 nucleoprotein to the number of nuclei in five fields of each slices at a 400 multiple signal magnification [29] (link).