To estimate the intracellular distribution of Nrf2 nucleoprotein, immunoflurescence analysis was perfomed as previously described [19 (link)]. Kidney tissues were embedded in paraffin, sectioned at 4μm, and then dewaxed in xylene and rehydrated with graded ethanol solutions. After protein blockade, the sections were incubated overnight at 4°Cwith the polyclonal Nrf2 primary antibody conjugated to FITC (dilution 1:150, Biorbyt, UK). Following washing triple in phosphate-buffered saline (PBS), slides were counterstained with 4’, 6-diamidino-2-phenylindole (DAPI) (Roche, Shanghai, China) to visualize the nuclei. The images were captured by a fluorescent microscope (Olympus U-25ND25, Tokyo, Japan) coupled to a digitial camera. The overlay color of blue staining in nucleus, accompanied with green staining both in cytoplasm and nucleus was considered to be positive. A semiquantitative numeric scores were determined for kidney samples based on the percentage of positive protein in five fields of each slices at a 400 multiple signal magnification using Image-pro plus software (Image pro plus 6.0, media Cybernetics, USA) [32 (link), 33 (link)].
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