Primary rabbit anti p65 antibody

Cells plated at 4×10⁵/well were harvested 0, 20 and 40 min after addition of 10 ng/ml TNFα the following day. Cells were washed once with 1× PBS followed by fixation for 20 min in 4% paraformaldehyde then 10 min in 0.3% H₂O₂ for permeabilization. Primary rabbit anti-p65 antibody (Abcam, Cambridge, MA, Cal# is 16502) was applied at 1∶800 dilution, cells were then incubated at 4°C overnight. Alexa Fluor 488 conjugated goat anti-rabbit secondary antibody (Invitrogen, Cal# is A11008) was then applied for 1 hr. Five pictures were taken from each well for analysis. To quantify the images, the fluorescence intensity of the nuclei and the fluorescence intensity of the whole cells were measured, and the proportion of total staining that was nuclear was determined.