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The antibody epitope was mapped to a site within a consensus tyrosine sulfation motif of CD162, previously shown to be essential for interaction with P selectin (and now shown to be essential for recognition of CD162 by L selectin).
ab78188 completely blocks recognition of CD162 by either P selectin or by L selectin, but does not affect leukocyte recognition of E selectin.
Our Abpromise guarantee covers the use of ab78188 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Blocking||Use at an assay dependent concentration.|
|WB||Use at an assay dependent concentration. Predicted molecular weight: 43 kDa.|
|IP||Use at an assay dependent concentration.|
|IHC-P||Use at an assay dependent concentration.
Indirect biotin streptavidin method recommended. Formalin fixed, deparaffinized sections were stained without pretreatment or microwaved for 15 minutes in 0.01 mol/L citrate buffer pH 6.0 before staining.
|IHC-Fr||Use at an assay dependent concentration.
Frozen sections were fixed in acetone for 10 minutes at 4 °C before staining.
|Flow Cyt||Use 0.01µg for 106 cells. ab170190-Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.|
Human peripheral blood lymphocytes stained with ab78188 (red line). Human whole blood was processed using a modified protocol based on Chow et al, 2005 (PMID: 16080188). In brief, human whole blood was fixed in 4% formaldehyde (methanol-free) for 10 min at 22°C. Red blood cells were then lyzed by the addition of Triton X-100 (final concentration - 0.1%) for 15 min at 37°C. For experimentation, cells were treated with 50% methanol (-20°C) for 15 min at 4°C. Cells were then incubated with the antibody (ab78188, 0.01μg/1x106 cells) for 30 min at 4°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) (ab150113) at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >30,000 total events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. Gating strategy - peripheral blood lymphocytes.
IHC image of CD162 staining in Human Normal aorta formalin fixed paraffin embedded tissue section, performed on a Leica BondTM 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 ab78188, 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.
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.