Anti-Glycophorin A + B antibody [HIR2] (ab15009)

ab15009 staining Human normal lung tissue. Staining is localised to cellular membranes. Left panel: with primary antibody at 1 µg/ml. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus, at RT. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 antigen retrieval buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins followed by blocking with Dako Protein block for 10 mins (containing casein 0.25% in PBS) then incubated with primary antibody for 20 mins and detected with Dako Envision Flex amplification kit for 30 mins. Colorimetric detection was completed with DAB for 5 mins. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Overlay histogram showing K562 cells stained with ab15009 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab15009, 1µg/1x106 cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in K562 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Flow cytometry analysis of human peripheral blood cells labelling Glycophorin A + B with ab15009. A APC-conjugated goat anti-mouse IgG was used as the secondary antibody.