IHC image of CD70 staining in human tonsil frozen tissue section, fixed in 10% paraformaldehyde (10 min). Staining was performed on a Leica Bond system using the standard protocol F. The section was then incubated with ab18619, 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.

Overlay histograms showing left Raji and right SHSY5Y cells stained with ab77868 (red line). The cells were incubated in 1x PBS / 10% normal goat serum to block non-specific protein-protein interactions followed by the antibody (ab77868) (1x10⁶ in 100µl at 1 µg/ml) for 30 min on ice.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor ^® 488, pre-adsorbed) (ab150177) was used at 1/2000 dilution for 30 min at 4°C. Isotype control antibody (black line) was mouse IgG1κ (ab170190) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

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

 

Immunohistochemical analysis of Human tonsil tissue, staining CD70 with ab77868.

Tissue was fixed with acetone and blocked with 6% serum for 30 minutes at 25°C. Samples were incubated with primary antibody (1/100 in 2% BSA in PBS) for 1 hour at 25°C. An AlexaFluor®488-conjugated goat anti-mouse monoclonal IgG (1/250) was used as the secondary antibody.

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ab77868 used in Flow Cytometry. Red histogram represents cells stained with secondary alone ab8517 at a 1/200 dilution, blue histogram - Formaldehyde fixed SK-RC-45 human renal carcinoma cells stained with ab78868 at a 1/100 dilution. FL1-H - green channel

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