Monoclonal anti NFKB p65 (Rel A) antibody was used to detect p65 by immunofluorescence at a dilution of 1:5000. HeLa cells were grown to sub-confluent on 18 mm2 glass coverslips #1.5. Cells were either unstimulated (A), or stimulated (B) with 50 ng/ml of TNF alpha for 30 min prior fixation. Cells were then fixed in methanol and blocked with 10% normal goat serum (NGS), in PBS, and Triton X 0.2% (Tx) and incubated for 1 hr at RT with primary ab, counterstained with DAPI and washed in PBS/NGS/Tx. Cells were incubated for 1 hr at RT with Atto 425 conjugated anti mouse secondary antibody for STED CW imaging. Data was collected on a STED-CW TCS-SP5 Confocal system equipped with a DFC 350FX camera allowing sequential acquisition in wide field, confocal and STED CW imaging on the same system.