Nrf2 antibody

The streptavidin-biotin complex immunohistochemical technique has been described previously (13 (link)). It was used to detect NRF2 protein in paraffin-embedded kidney tissue sections by permeabilizing with 0.3% Triton X-100 (cat. no. P0096; Beyotime Institute of Biotechnology) at room temperature for 10 min, then blocked with 10% goat serum (cat. no. C0265; Beyotime Institute of Biotechnology) at 37˚C for 10 min, incubated overnight at 4˚C with 1:400 NRF2 antibody (cat. no. ab92946; Abcam), incubated 30 min at 37˚C with 1:500 Biotin-labeled secondary antibody (cat. no. A0277; Beyotime Institute of Biotechnology), incubated 1 h at room temperature with the 1:400 Streptavidin-HRP (cat. no. A0303; Beyotime Institute of Biotechnology) and dyed 2-5 min at room temperature with DAB + 30% H₂O₂. Positive expression in the cytoplasm and/or nucleus was stained brown (original magnification, x200; Olympus BX50; Olympus Corporation). The optical density of positive staining was semi-quantitatively evaluated using Image Pro^®plus version 6.0 software (Media Cybernetics, Inc.).

Paraffin sections were dewaxed and hydrated, and cell slides were fixed in 4% paraformaldehyde. The sections were incubated in 0.1% triton X-100 (Beyotime) for 30 min and in goat serum for 15 min. Then, the sections were incubated with Nrf2 antibody (1:200) at 4 °C overnight, and with Cy3-labeled goat-anti-rabbit IgG (1:200, Beyotime) at 37 °C for 1 h. DAPI (Beyotime) was added dropwise into the sections. The sections were then washed with phosphate buffered solution buffer and added with anti-fluorescence quencher (Solarbio) to slow down the fluorescence quenching speed. The staining was observed under a fluorescence microscope (Olympus) at 400× magnifications.