Anti-E Cadherin antibody [M168] - C-terminal (ab76055)

Immunohistochemistry staining of E Cadherin in human MMCR (Muller’s muscle conjunctival resection) tissue.

Xylene and rehydration with serial ethanol dilutions were used for deparaffinization. Slides were washed twice for 5 minutes in 0.25% Triton X-100 for permeabilization and blocked for 2 hours at room temperature with 2% BSA and 10% normal donkey serum in PBS. Slides were incubated overnight at 4°C with ab76055. The next day, the slides were washed twice for 5 minutes in PBS and incubated for 1–2 hours with respective secondary antibodies diluted in blocking solution (1:200–800). Vecta shield mounting medium with 4′,6-diamidino-2-phenylindole (DAPI) was placed over the slides and covered with a glass coverslip. Slides were analyzed using the Zeiss LSM 710 Confocal Microscope.

Blocked and probed in the presence of 5% milk, and the image was captured using chemiluminescence and film.

IHC image of E Cadherin staining in human colon 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, for 20 minutes. The section was then incubated with ab76055, 5 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.

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.

ICC/IF image of ab76055 stained A431 (Human epidermoid carcinoma cell line) cells.

The cells were 4% formaldehyde fixed (10 minutes) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab76055, 1/100 dilution) overnight at +4°C. The secondary antibody (green) was ab96879, DyLight^® 488 goat anti-mouse IgG (H+L) used at a 1/250 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

Overlay histogram showing A431 (Human epidermoid carcinoma cell line) cells stained with ab76055 (red line).

The cells were fixed with 4% paraformaldehyde (10 minutes) and then permeabilized with 0.1% PBS-Tween for 20 minutes. 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 (ab76055, 1/100 dilution) for 30 minutes at 22ºC. The secondary antibody used was a 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 IgG1 [ICIGG1] (ab91353, 2 µg/1x10⁶ cells) used under the same conditions.

Acquisition of >5,000 events was performed.

This antibody gave a positive signal in A431 cells fixed with 80% methanol (5 minutes)/permeabilized with 0.1% PBS-Tween for 20 minutes used under the same conditions.