4.8. Observing p65 Nuclear Translocation by IF

Nuclear translocation of NF-κB (p65) was analyzed by immunofluorescence. The cells were cultured in a 6-well plate pre-installed with sterile cell slides, and the cells were treated according to the experimental design. The medium was discarded and fixed with 4% paraformaldehyde for 15 min at room temperature. PBS washed three times. Blocked with 1 × PBS containing 5% goat serum, 0.3% Triton X-100 for 1 h at room temperature. The primary antibody against p65 and secondary antibody rabbit anti-goat IgG/FITC were diluted with 1 × PBS containing 1% BSA, 0.3% Triton X-100. The cells were incubated with primary antibody overnight at 4 °C, followed by incubation with rabbit anti-goat IgG/FITC antibody. Then, the cells were washed by PBS three times. Nuclei were stained with DAPI. After washing with PBS, the slides were mounted with anti-fluorescence quenching mounting medium, and the nuclear entry of p65 was observed under a confocal laser microscope.