Overlay histogram showing A431 cells stained with ab77684 (red line). The cells were fixed with 4% paraformaldehyde (10 min)) and then permeabilized with 0.1% PBS-Triton for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab77684, 1µg/1x10⁶ cells) for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG3 FITC (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in A431 cells fixed with 80% methanol/permeabilized in 0.1% PBS-Triton used under the same conditions.

IHC image of ab77684 staining in human skin formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab77684, neat, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.