Nuclear dye dapi

The mice were divided into the model (DSS), experimental (DSS + DGBX), and control (normal saline) groups, with each group consisting of 6 mice. On the day before each specific time point (days 3, 5, and 9), bromodeoxyuridine (BrdU) with a concentration of 1 mg/mouse was injected into the mice intraperitoneally. After 24 h, the colonic tissue of the mice was washed with PBS and placed in an embedding device with an optimal cutting temperature (OCT) compound. The embedded tissue was directly frozen in a refrigerator at −80°C. The tissue was sliced into sections measuring 10 μm, fixed with 4% paraformaldehyde for 10 min, and sealed with an immunostaining blocking solution for 60 min. BrdU dyeing was conducted as instructed (Sigma-Aldrich, St. Louis, USA). The BrdU antibody was added based on the concentration of 1 : 100, incubated overnight at 4°C, and washed with PBS three times for 5 min each time. Subsequently, the Cy3-conjugated antibody was incubated at 37°C for 30 min. After PBS cleaning, staining with a nuclear dye DAPI (Beyotime Biotechnology, Shanghai, China) for 10 min was conducted. Fluorescence was observed and photographed using an Olympus confocal microscope (BX53 microscope, Olympus, Japan).

AML12 cells were grown and treated on cell culture dish with glass bottom. After the treatment, the cells were washed by PBS, fixed by paraformaldehyde, blocked by the goat serum and were incubated with primary antibody (Nrf2, rat monoclonal, CST, #14596, 1:50) at 4°C overnight. The cells were subsequently incubated with the secondary antibody (Alexa Fluor 488 Donkey anti‐Rat IgG (H + L), Invitrogen, A21208) for 2 hours and the nuclear dye DAPI (Beyotime) for 15 minutes at room temperature. Fluorescent images were obtained by confocal microscope (Nikon), and statistical analysis was performed according to the previous study.²²