Nrf2 Immunofluorescence Imaging Protocol

Cells (5 × 10⁵ per well) were seeded into six well plates containing a coverslip. The next day, cells were rinsed with ice-cold PBS and fixed with 4% paraformaldehyde for 10 min at room temperature, followed by permeabilization with 0.1% sodium citrate with 0.1% Triton X-100. The cells were incubated with Nrf2 antibody at 1:1000 dilution (12721S; Cell Signaling, Danvers, MA, USA) for 1 h at room temperature. The cells were then washed with cold PBS three times (5 min each) and incubated with Alexa 514-labeled anti-rabbit secondary antibody (1:1000) (A11061; Thermo Fisher Scientific, Waltham, MA, USA) at room temperature for 1 h. The cells were then incubated with conjugated actin antibodies for 15 min. After washing with PBS, the coverslips were mounted onto the slides with ProLong Diamond Antifade Mountant with DAPI (P36962; Thermo Fisher Scientific, Waltham, MA, USA). Standard immunofluorescence methods were used as described previously [28 (link)]. Imaging was performed using EvosFL fluorescence microscope (Life Technologies, Carlsbad, CA, USA) at 40× objective magnification. Fluorescence intensities from images of six randomly selected microscopic fields of cells were semi-quantitatively analyzed by densitometry (ImageJ software, NIH).