IHC image of CD200R staining in a section of frozen normal mouse spleen performed on a Leica BOND^TM system using the standard protocol F.

The section was fixed with 10% paraformaldehyde for 10 mins prior to staining. The section was then incubated with ab243839, 5 µg/ml, for 15 mins at room temperature. A rabbit anti-rat bridger antibody, ab102248, was added at a 1/500 dilution for 8 mins at room temperature and then 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.

Flow cytometry overlay histogram showing left control cells and right C57 BL/6 mouse splenocytes treated with 10 ug/ml LPS for 3 days stained with ab243839 (red line). The cells were incubated in 1x PBS containing 10% mouse serum and 10% normal goat serum followed by the antibody (ab243839) (1x10⁶ in 100 μl at 1 μg/ml) for 30 min on ice.

The secondary antibody Goat anti-rat IgG H&L (Alexa Fluor ^® 488, pre-adsorbed) (ab150165) was used at 1/2000 for 30 min on ice.

Isotype control antibody (black line) was Rat IgG1κ (ab18407) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. Events were gated on live CD19 positive B cells.