Validation of Integrin α6/CD49f to identify CRC cells in patient samples.

Freshly harvested tumor or normal tissue was snap frozen and banked at −80°C. A gastrointestinal pathologist confirmed the histopathology diagnosis of each specimen independently. Normal tissue was obtained from a site distal from the primary colon tumor. Fresh frozen tissues were sectioned at a 6 µm thickness. Slides were fixed with 4% paraformaldehyde, air-dried, and stored at −20°C until use. After treatment with 10% normal goat serum and 0.1% Triton X-100 for 45 min, slides were incubated with monoclonal affinity purified mouse anti-human EpCAM (ab20160, Abcam) at a final dilution of 1∶200.

Panels shown are EpCAM (ab20160, 1/200 dilution) and merge for normal tissue.

Overlay histogram showing DU 145 (Human prostate carcinoma cell line) cells stained with ab20160 (red line).

The cells were fixed with methanol (5 min) 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 (ab20160, 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 slightly decreased signal in DU 145 cells fixed with 4% paraformaldehyde (10 min) used under the same conditions.

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

IHC image of ab20160 staining in human breast carcinoma 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 mins. The section was then incubated with ab20160, 10µg/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 hematoxylin 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.