Rabbit anti nf κb p65 primary antibody

IMG cells were grown on poly-D-lysine-coated coverslips and then treated as described in the figure legend. After treatments, the cells on coverslips were rinsed with PBS and fixed for 15 minutes with 4% paraformaldehyde at room temperature. After fixation, cells were rinsed with PBS and then permeabilized at room temperature for 5 minutes in PBS solution containing 0.5% Triton X-100. Cells were then blocked for 1 hour at room temperature in PBS solution containing 5% normal goat serum, 0.5% Triton X-100, 1% bovine serum albumin and 0.3M glycine. Cells were then incubated overnight at 4°C with a rabbit anti-NF-κB/p65 primary antibody (Cell Signaling Technology, Cat. #8242, RRID:AB_10859369, 1:500) diluted in blocking buffer. The next day the cells were washed with PBS, incubated with Alexa Fluor 488-conjugated anti-rabbit antibody (Invitrogen, Cat. #32731; 1:1000) for 1 hour at room temperature. After secondary antibody incubation, cells were washed with PBS and mounted onto glass slides using a fluorescent mounting medium (DakoCytomation, Cat. #S3023). Images were obtained using a Zeiss Cell Observer spinning disc confocal microscope system (Carl Zeiss, Germany). ImageJ software (NIH, Bethesda, MD) was used to analyze images.