Nrf2 Immunostaining in Tissue Sections

Immunohistochemistry and immunofluorescent staining were performed as previously described [52 (link)]. In detail, the tissue sections (4 mm) were incubated with Nrf2 antibody, (Sigma, St. Louis, MO, USA), which was diluted in Antibody Diluent Reagent Solution (Invitrogen, Carlsbad, CA, USA) and reactions were carried out overnight at 4 °C. After washing with PBS for three times (5 min each time), the sections were incubated with horse anti-mouse antibody (Sigma, Louis, MO, USA) at room temperature for 30 min. After washing three times with PBS, the slides were stained with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI; Beyotime Biotech Inc., Nantong, China) for 2 min to show the locations of nuclei. Images were acquired using an Olympus BX40 microscope (Olympus Corporation, Tokyo, Japan). The Image-pro Plus 5.0 (Media Cybernetics, Inc., Rockville, MD, USA) was applied to calculate the expression of Nrf2.