Immunostaining of NF-κB p65 Nuclear Translocation

Cells were grown on glass coverslips in 24-well plate and fixed in 4% paraformaldehyde for 15 min, permeabilized with 0.1% Triton X-100 in PBS in room temperature for 20 min, followed by 3% BSA blocking for 30 min. NF-κB p65 (CST, Danvers, MA) was diluted in 3% BSA/PBS at 1:00 and incubated overnight. The NF-κB p65 antibody was detected with anti-rabbit IgG secondary antibody Alexa Fluor 594 (ThermoFisher Scientific, Waltham, MA). The coverslips were mounted onto microscope slides in Vectashield mounting medium for fluorescence containing DAPI (Vector Laboratories, Burlingame, CA) and fluorescent images were visualized using Leica TCS II Confocal Microscope System.