Recombinant Anti-CD62P antibody [AK-6] (ab6632)

Flow cytometry staining of human whole blood with ab6632 (right) or mouse IgG1κ (ab170190) isotype (left). Red blood cells of 200 µl blood were lysed, then cells were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10% normal goat serum to block Fc receptors and non-specific protein-protein interaction followed by the antibody (ab6632) or mouse IgG1κ (ab170190) isotype (1x10⁶ 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 CD42a PE. Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. Events were gated on alive neutrophils.

IHC image of CD62P staining in a section of frozen normal human colon* performed on a Leica BOND™ system using the standard protocol. The section was fixed in 10% paraformaldehyde (10 min) prior to staining. The section was incubated with ab6632, 0.01ug/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 the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

IHC image of CD62P staining in a section of formalin-fixed paraffin-embedded normal human bone marrow* 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 ab6632, 10ug/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 the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

IHC image of CD62P staining in a section of formalin-fixed paraffin-embedded normal human colon* 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 ab6632, 10ug/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 the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre

ab6632 staining CD62P in Human whole blood. The cells were fixed with 4% paraformaldehyde (10 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab6632 at 10μg/ml and ab6046, Rabbit polyclonal to beta Tubulin - Loading Control. Cells were then incubated with ab150117, Goat polyclonal Secondary Antibody to Mouse IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and ab150080, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor® 594) at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).