4.12. Immunofluorescence Staining of Nrf2 Nuclear Translocation

Nuclear translocation of Nrf2 was examined by immunofluorescence staining. HUVECs were fixed with 3.7% paraformaldehyde in PBS for 15 min followed by treating permeabilization buffer (0.1% Triton X-100 in PBS) for 10 min. HUVECs were incubated with blocking solution (2% bovine serum albumin) for 30 min at room temperature. After washing with PBS, HUVECs were treated with anti-Nrf2 antibody (1:200 dilution, Santa Cruz Biotechnology) at 4 °C for overnight. The secondary antibody labeled with Alexa Fluor^® 488 (1:500 dilution, Santa Cruz Biotechnology, Dallas, TX, USA) was treated for 1 h. Nuclei were counterstained with 2 μg/mL Hoechst 33342 for 10 min. The stained HUVECs were visualized using a fluorescence microscope.