Overlay histogram showing HAP1 wildtype (green line) and HAP1-CD81 knockout cells (red line) stained with ab79559. The cells were fixed with 4% formaldehyde (10 min)  and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab79559, 1µg/ml) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-mouse IgG (H&L) presorbed (ab150117) at 1/2000 dilution for 30 min at 22°C.

A mouse IgG1 isotype control antibody  (ab170190) was used at the same concentration and conditions as the primary antibody (HAP1 wildtype - black line, HAP1-CD81  knockout - grey line). Unlabelled sample was also used as a control (this line is not shown for the purpose of simplicity).

Acquisition of >5,000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter.

This antibody can also be used in HAP1 cells fixed with  80% methanol (5 min), , permeabilized with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

IHC image of CD81 staining in Human normal liver formalin fixed paraffin embedded tissue section, 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 20 mins. The section was then incubated with ab79559, 5 µg/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.

Overlay histogram showing MOLT4 cells stained with ab79559 (red line). The cells were fixed with 4% paraformaldehyde and incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab79559, 1µg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MOLT4 cells fixed with methanol (5 min) used under the same conditions.

Please note that Abcam do not have data for use of this antibody on non-fixed cells. We welcome any customer feedback.

Anti-CD81 antibody [M38] (ab79559) at 2 µg/ml + Jurkat (human T cell leukemia cell line from peripheral blood) whole cell lysate

Secondary
IRDye800-conjugated Anti-Mouse Secondary Antibody

Performed under non-reducing conditions.

Predicted band size: 25 kDa
Observed band size: 25 kDa

Flow cytometry analysis using human peripheral blood cells with ab79559.