Fitc conjugated goat anti rabbit igg

Cells were rinsed twice with cold phosphate-buffered saline (PBS, Beijing Zhongshan Golden Bridge Biotechnology, Beijing, China) and fixed with 4% paraformaldehyde at room temperature for 15 min. Subsequently, cells were washed three times (5 min each) in PBS, permeated by incubating in 0.05% Triton X-100 for 10 min (Beijing Zhongshan Golden Bridge Biotechnology, Beijing, China), washed again, and blocked with 10% normal goat serum blocking solution for 1 h at 37°C (Beijing Zhongshan Golden Bridge Biotechnology, Beijing, China). Then, cells were incubated overnight at 4°C with antibody anti-Nrf-2 (1:100; Santa Cruz Biotechnology, CA, USA). Cells were then washed three times with PBS (5 min each) before incubation with FITC-conjugated goat anti-rabbit IgG (1:50; Beijing Zhongshan Golden Bridge Biotechnology Co. Ltd., Beijing, China) for 1 h in humidified chambers at 37°C. The cell nucleus was stained with PI (Beyotime Institute of Biotechnology, Jiangsu, China). The slides were washed three times with PBS and mounted in PBS/glycerol (50:50). In the negative control group, the primary antibody was omitted while the other steps remained the same; no fluorescence signals were detected. Images were captured on a Leica TCS SP2 confocal laser imaging system (Leica Microsystems, Wetzlar, Germany). All immunocytochemical experiments were repeated three times.