Immunofluorescence Analysis of Nrf2 in IPEC-J2 Cells

IPEC-J2 cells were cultured in a 24-well plate. Cells were treated as described for western blot analysis. The methods used for immunofluorescence staining of cells were described previously [31 (link)] and were followed with slight modifications. In brief, after 30 min of fixation with 4% paraformaldehyde solution, cells were permeabilized with 0.2% Triton X-100 for 10 min and blocked with 5% BSA for 1 h. Cells were incubated with a rabbit anti-Nrf2 antibody overnight at 4 °C and were then incubated with an Alexa Fluor 594-conjugated secondary antibody (ZF-0513, ZSGB-BIO) for 1 h. Subsequently, cell nuclei were stained with DAPI (Alexa Fluor® 555; ab150078; Abcam) for 10 min and imaged immediately using an Olympus fluorescence microscope (Tokyo, Japan). Each treatment group was analyzed in triplicate.