Nrf2 primary antibody

To examine the efficiency of CUR on NRF2 nuclear translocation, MG-63 cells were treated with CUR in a dark-wall 96-well plate (1.0 × 10⁴ cells/well) for 48 h. After that, cells were gently washed with cold PBS and fixed by 4% formaldehyde for 10 min. The fixed cells were permeabilized by 0.05% Triton X-100 for 5 min at room temperature. Thereafter, BlockAid™ Blocking Solution was used to minimize the unspecific binding of the antibody. Afterwards, NRF2 primary antibody (Abcam, Cambridge, UK) was diluted (1:50) in blocking buffer and added into the well at 4 °C overnight. On the second day, the primary antibody was washed away with cold PBS for 3 × 10 min and cells were incubated for 1 h at room temperature in the dark with the secondary antibodies (Abcam, Cambridge, UK). A total of 1 μg/mL Hoechst 33342 was used to stain the nuclei of MG-63 cells. Finally, cells were rinsed with cold PBS. Images were acquired using a fluorescence microscope.

Colonic tissue sections (5 μm) were washed in 10% PBS for 20 min, blocked with 10% goat serum, and then incubated at 4°C overnight with Nrf2 primary antibody (Abcam, Cambridge, UK) in PBS containing 1% BSA (1 : 50). After washing with PBS, the tissues were incubated at 37°C for 2 h with rhodamine-labeled rabbit IgG antibody (Santa Cruz Biotechnology, Santa Cruz, CA). Then, the slides were stained with the fluorochrome dye DAPI (Santa Cruz Biotechnology, Santa Cruz, CA). The laser scanning confocal microscope (Olympus FluoView FV1000, Japan) was used to visualize Nrf2 and the cell nuclei with peak excitation wavelengths of 570 and 340 nm [27 (link)].