Lewis rat splenocytes stained with ab243837 (right) or mouse IgG2a kappa (ab170191) isotype (left). Cells were incubated for 30 min on ice in 1x PBS containing 10 % rat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab243837) or mouse IgG2a kappa (ab170191) isotype (1x 10⁶ in 100 µl at 0.2 µg/ml) for 30 min on ice.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^®488, pre-adsorbed) (ab150117) was used at 1/2000 dilution for 30 min on ice. The cells were simultaneously stained with CD3.

Acquisition of >30,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. Events were gated on live single cells.

IHC image of CD2 staining in a section of formalin-fixed paraffin-embedded normal rat spleen* performed on a Leica BOND™ 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 20mins. The section was then incubated with ab243837, 5µg/ml, 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. 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.

*Tissue obtained from Charles River.