ab30470 staining CD45 in Jurkat (Human T cell leukemia cell line from peripheral blood) cells. The cells were fixed with 4% formaldehyde (10 minutes), then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour. The cells were then incubated overnight at +4°C with ab30470 at 5 µg/ml (shown in green) and ab206369, Rabbit monoclonal to beta Tubulin (Alexa Fluor^® 594), at 1/250 dilution (shown in red). This was followed by an incubation at room temperature for 1 hour with ab150117, Goat Anti-Mouse IgG H&L (Alexa Fluor^® 488) preadsorbed, at 1 µg/ml (shown in green). Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

This product also gave a positive signal under the same testing conditions in Jurkat cells fixed with 80% methanol (5 minutes).

Flow cytometry staining of human whole blood with ab30470 (right) or mouse IgG2aκ; (ab18413) 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 (ab30470) or mouse IgG2aκ; (ab18413) 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.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 530/30 bandpass filter. Events were gated on alive cells.

IHC image of CD45 staining in human tonsil formalin fixed paraffin embedded tissue section, performed on a Leica Bond^TM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab30470, 1 µg/ml, for 15 minutes 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.

Overlay histogram showing Jurkat (Human T cell leukemia cell line from peripheral blood) cells stained with ab30470 (red line). The cells were fixed with 4% paraformaldehyde (10 minutes) 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 (ab30470, 1 µg/1 x 10⁶ cells) for 30 minutes at 22ºC. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 minutes at 22ºC. Isotype control antibody (black line) was mouse IgG2a [ICIGG2A] (ab91361, 1 µg/1 x 10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Jurkat cells fixed with 80% methanol (5 minutes) used under the same conditions.

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