IHC image of BRCA1 staining in a section of formalin-fixed paraffin-embedded normal human breast* 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 (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab16781, 5ug/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

ab16781 staining BRCA1 in MCF7 cells. The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 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 with ab16781 at 1μg/ml concentration and ab6046 at 1/1000 dilution overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Mouse IgG H&L (ab150117) at 1/1000 dilution (shown in green) and Goat anti-Rabbit IgG (ab150080) at 1/1000 dilution (shown in pseudocolor red). Nuclear DNA was labelled in blue with DAPI.

Overlay histogram showing MCF7 cells stained with ab16781 (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 (ab16781, 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 IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in MCF7 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

ab16781 (1/200) detecting BRCA1 in HeLa cells in conjunction with a goat anti-mouse secondary antibody conjugated to Cy3 (red). Cells were also counterstained with DAPI in order to highlight the nucleus (blue), treated with Bleomycin and incubated with an antibody against Histone H2AX in order to create and expose DNA double strand breaks (green). Please refer to abreviews for further details.

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