Lewis rat bone marrow cells stained with ab6331 (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 (ab6331) 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 Ter-119.

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 Transferrin receptor staining in a section of formalin-fixed paraffin-embedded normal rat placenta performed on a Leica BOND™ system using the standard Protocol F, omitting the post-primary step. The section was pre-treated using heat mediated antigen retrieval with EDTA buffer (pH9, epitope retrieval solution 2) for 20mins. The section was then incubated with ab6331, 5µg/ml, for 15 mins at room temperature. The section was then incubated with ab98792 (Rabbit polyclonal Secondary Antibody to Mouse IgG - H&L, pre-adsorbed) 1/500 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 hematoxylin 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.

IHC image of Transferrin receptor staining in normal mouse placenta formalin fixed paraffin embedded tissue section, using Mouse on Mouse detection kit ab127055. The section was pre-treated using pressure cooker heat mediated antigen retrieval with sodium citrate buffer (pH6) for 30mins. The section was incubated with ab6331, 5µg/ml overnight at +4°C. The section was counterstained with hematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.