Immunofluorescent staining of A549 (ATCC CCL-185) cells. Cells were seeded in a 96 well imaging plate at ~ 10 000 cells per well. After overnight incubation, cells were either mock treated (media, left) or exposed to TNF (20ng/ml, right) for 15 minutes. After treatment cells were stained using the Triton™ X-100 perm protocol and the anti-NF-kappaB antibody. The second step reagent was Alexa-Fluor® 488 goat anti-mouse IgG (Invitrogen). The image was taken on a BD Pathway™ 855 Bioimager with a 20x objective. This antibody also stains U-2 OS (ATCC HTB-96) and HeLa (ATCC CCL-2) cells and can be used with either perm protocol.