Recombinant Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

Blocking and diluting buffer: 5% NFDM/TBST

Multi-bands can refer to PMID: 11212238; PMID: 14695147 and PMID: 22659456

PMA induced cell fusion, DYSF expression, and activation of PKC in BeWo cells while 4αPMA was inactive

Immunofluorescence analysis of BeWo cells treated with 0.25% DMSO (controls), 10 nM PMA, or 10 nM 4αPMA for 72 h. The cells were then fixed and subsequently double-labeled for detection of DYSF (red) and E-cadherin (green). Nuclei were labeled with DAPI. While there can be a low level of spontaneous fusion in control cells (in our hands this ranges from about 4 to 9%), most cells are not fused and have at their borders intact E-cadherin labeling. Moreover, DYSF labeling was not detectable in non-fused BeWo cells. However, treatment of BeWo cells with 10 nM PMA for 72 h led to increased levels of cell fusion as indicated by the breakdown of E-cadherin labeling and the expression of DYSF in fused cells. When BeWo cells were treated with 10 nM 4αPMA for 72 h there was no detectable increase in cell fusion or DYSF expression. Arrows indicate areas enlarged and placed in insets. Bar = 50 µm.

Mesenchymal cancer cells show increased metastasis while not requiring MET for solid tumor formation.

ZEB1 or E-cadherin staining of metastases in ICI-mice. Note the higher E-cad and lower ZEB1 expression in the metastatic cells expressing OVOL1 or ZEB1-shRNA (sh4). Scale bar represents 100 µm.

ab40772 staining E Cadherin in HT-29 (Human colorectal adenocarcinoma) cells by ICC/IF (Immunocytochemistry/Immunofluorescence).  Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. Samples were incubated with primary antibody at 1/500 dilution. An Alexa Fluor^® 488 Goat anti-Rabbit (ab150077) was used as the secondary antibody at 1/1000 dilution. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (ab195889) at 1/200 dilution was used as a counterstain. DAPI was used as a nuclear counterstain. This is a confocal image showing membranous staining on HT-29 cell line.

Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling E Cadherin with purified ab40772 at 1:30 dilution (10 µg/ml) (red). 10⁶ cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

For organotypic cultures, frozen sections were permeabilized with 80% MeOH for 5 min. at 4°C and acetone for 2 min. at −20°C prior to staining. Nonspecific staining was blocked by 10% goat serum. Sections were incubated for one hour in a PBS solution containing ab40772 (1/400). The primary antibody were visualized by staining one hour with goat anti-rabbit Alexa 488. Nuclei were counterstained with 30-minute incubation with Hoechst stain (1/2000).

Normal human dermal fibroblasts and Type 1 Collagen were mixed, polymerized and cultured After allowing 2 days for the fibroblasts to contract and reorganize the tissue, K-HME cells were plated on the upper surface at a concentration to provide a confluent monolayer of cells. Culture continued for 2 days submerged in keratinocyte medium with 20 μg/ml ascorbic acid. For air/liquid interface culture, tissues were transferred to clear Transwell 6-well inserts and placed into deep-well dishes for the remainder of the culture period.

Fluorescent immunohistochemical analysis of paraffin-embedded human breast carcinoma tissue using unpurified ab40772. Green-E-Cadherin red-PI.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Overlay histogram showing A431 (Human epidermoid carcinoma cell line) cells stained with unpurified ab40772 (red line). 10⁶ cells were fixed with 80% methanol (5 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 (ab40772, 1/1000 dilution) for 30 minute at 22°C. The secondary antibody used was DyLight^® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 minutes at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1 μg/1x10⁶ cells) used under the same conditions. Unlabeled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Formalin/PFA-fixed paraffin-embedded human colonic adenocarcinoma tissue stained for E Cadherin with unpurified ab40772 at a 1/500 dilution in immunohistochemical analysis.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Exposure time: 3 minutes for ab40772 and ab133597, 32 seconds for GAPDH.

Blocking and diluting buffer: 5% NFDM/TBST

Multi-bands can refer to PMID: 11212238; PMID: 14695147 and PMID: 22659456

Immunocytochemistry/Immunofluorescence analysis of MCF7 (human breast adenocarcinoma epithelial) cells labeling E Cadherin with ab40772. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% tritonX-100. Samples were then incubated with the primary antibody at a 1/500 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at a 1/1000 dilution (green). The nuclear counter stain is DAPI (blue). Counterstained with ab195889 anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at a 1/200 dilution (red).

Confocal image shows membranous staining on MCF7 cell line.

Formalin-fixed, paraffin-embedded human lung adenocarcinoma tissue stained for E Cadherin with unpurified ab40772 at a 1/500 dilution in immunohistochemical analysis.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Exposure time: 1 second for ab40772, 3 minutes for ab133597, 32 seconds for GAPDH

Blocking and diluting buffer: 5% NFDM/TBST

Multi-bands can refer to PMID: 11212238; PMID: 14695147 and PMID: 22659456

Formalin-fixed, paraffin-embedded human breast carcinoma tissue stained for E Cadherin with unpurified ab40772 at a 1/500 dilution in immunohistochemical analysis.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Blocking and diluting buffer and concentration 5% NFDM/TBST.

The full-length of E-cadherin is 120 kDa. The other bands are due to proteolytic cleavages in different Cadherin domains. (Ref: PMID: 14695147)

Formalin-fixed, paraffin-embedded human papillary carcinoma of thyroid gland tissue stained for E Cadherin with unpurified ab40772 at a 1/500 dilution in immunohistochemical analysis.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Formalin-fixed, paraffin-embedded human transitional cell carcinoma of kidney tissue stained for E Cadherin with unpurified ab40772 at a 1/500 dilution in immunohistochemical analysis.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunohistochemistry of breast carcinoma staining E Cadherin with ab40772 at 1μg/ml

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Produced using unpurified ab40772

Equilibrium disassociation constant (K_D)
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