Overlay histogram showing Daudi cells stained with ab213318 (red line).The cells were incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab213318, 1/10 dilution) for 30 min at  4°C. The secondary antibody (black line) used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150081) at 1/2000 dilution for 30 min at 4°C. Unlabelled sample (blue line) was also used as a control.

Acquisition of >30,000 total events were collected.

IHC image of CD27 staining in human normal tonsil formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol B. 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 ab213318, 1µg/ml, for 15 mins at room temperature. A goat anti-rabbit biotinylated secondary antibody was used to detect the primary, and visualized using an HRP conjugated ABC system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.

 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