Fluorescein isothiocyanate conjugated anti mouse secondary antibody

Immunofluorescence assays were performed as published (51 (link),52 (link)). Mouse anti-γ-H2AX (Ser139, clone JBW301, Millipore) was used at 1:500 dilution. The monoclonal anti-BrdU antibody (BD Biosciences) was used at 1:200 concentration. The secondary antibody, goat–anti-mouse immunoglobulin G (IgG) Alexa fluor 594 or fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (sigma) was used at 1:500 dilution. The slides were viewed at 1000× magnification on an NIKON 90i fluorescence microscope (photometric cooled mono CCD camera).

HaCaT cells (3 × 10⁵ cells/mL/well) were seeded into an eight-well chamber containing DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) P/S and incubated at 37 °C for 16 h. After treatment with CSK or H₂O₂, the cells were fixed using a 4% formaldehyde solution and permeabilized using 0.2% TritonX-100 (Sigma, St. Louis, MO, USA). Subsequently, the cells were blocked with 3% bovine serum albumin and then incubated with an anti-Nrf2 antibody (1:50 in 3% bovine serum albumin) for 2 h at room temperature. Then, fluorescein isothiocyanate-conjugated anti-mouse secondary antibody (Sigma, St. Louis, MO, USA) was added, and the cells were incubated for 2 h at room temperature. The cells were mounted onto the cover glass, using ProLong Gold Antifade Mountant with DNA Stain DAPI (Thermo Fisher, Waltham, MA, USA), and observed and photographed using a confocal microscope (Leica, Wetzlar, Germany). The exposure time was consistent across samples in all experiments.