Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker

28 product images

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

  Blocking and diluting buffer and concentration: 5% NFDM/TBST.
  
  Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 was as GAPDH loading control.
  
  Exposure time: Lane1: 3 seconds; Lane 2-5: 40 seconds.
  
  A375, HeLa and HT-1080 were reported as negative or express low level of E cadherin (PMID: 30393081, PMID: 16980628, PMID: 34715746), PMID: 25411788).

  All lanes: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/1000 dilution

  Lane 1: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 2: HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 3: A375 (Human malignant melanoma epithelial cell) whole cell lysate at 20 µg

  Lane 4: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Lane 5: HT-1080 (Human fibrosarcoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 97 kDa

• 

  Image from Roca H. et al PLoS One. 2013 Oct 4;8(10):e76773. doi: 10.1371/journal.pone.0076773. eCollection 2013.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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.

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  Immunocytochemistry/ Immunofluorescence - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  **Benchmark:** ab40772 (recombinant rabbit monoclonal antibody). The data in this figure was generated using ab40772. This antibody was used as a reference for comparison with Anti-E Cadherin antibody [36/E-Cadherin] ab287970. The nuclear counter stain is DAPI (blue).
  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized MCF7 (Human breast adenocarcinoma cell line) whole cell lysate labeling E Cadherin with ab40772 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) secondary antibody at 1/1000 dilution (green). Confocal image showing membranous staining in MCF7 cell line. Tubulin is detected with Anti-alpha Tubulin mouse MAb (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889) at 1/200 dilution (red).
  **Negative control: **MDA-MB-231 (PMID: 10545506).

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  Western blot - 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

  All lanes: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/10000 dilution

  Lane 1: MCF7 (Human breast adenocarcinoma epithelial cell). Whole cell lysates at 20 µg

  Lane 2: HT-29 (Human colorectal adenocarcinoma epithelial cell). Whole cell lysates at 20 µg

  Lane 3: PC-3 (Human prostate adenocarcinoma epithelial cell) Whole cell lysates at 20 µg

  Lane 4: MDA-MB-231 (Human breast adenocarcinoma epithelial cell) Whole cell lysates (negative control) at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Performed under reducing conditions.

  Predicted band size: 97 kDa

  Exposure time: 23s

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  Immunocytochemistry/ Immunofluorescence - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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 (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) was used as the secondary antibody at 1/1000 dilution. Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) (Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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.

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

  Exposure time: 3 minutes for ab40772 and Anti-E Cadherin antibody [EPR699] ab133597, 32 seconds for GAPDH.
  

  Blocking and diluting buffer: 5% NFDM/TBST

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

  Lane 1: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/5000 dilution

  Lane 1: Western blot - Anti-E Cadherin antibody [EPR699] (Anti-E Cadherin antibody [EPR699] ab133597) at 1/5000 dilution

  Lane 2: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/2000 dilution

  Lane 2: Western blot - Anti-E Cadherin antibody [EPR699] (Anti-E Cadherin antibody [EPR699] ab133597) at 1/2000 dilution

  Lane 3: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  Lane 3: Western blot - Anti-E Cadherin antibody [EPR699] (Anti-E Cadherin antibody [EPR699] ab133597)

  All lanes: PC-3 (Human prostate adenocarcinoma epithelial cell) Whole cell lysates at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 97 kDa

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  Image from Omata W. et al PLoS One. 2013 Nov 13;8(11):e81003. doi: 10.1371/journal.pone.0081003. eCollection 2013.

  Immunocytochemistry/ Immunofluorescence - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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.

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  Flow Cytometry (Intracellular) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  Intracellular Flow Cytometry analysis of MCF7 (Human breast adenocarcinoma epithelial cell) cells labeling E Cadherin with purified ab40772 at 1/30 dilution (10μg/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).
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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.

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

  Exposure time: 1 second for ab40772, 3 minutes for Anti-E Cadherin antibody [EPR699] ab133597, 32 seconds for GAPDH
  

  Blocking and diluting buffer: 5% NFDM/TBST

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

  Lane 1: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/5000 dilution

  Lane 1: Western blot - Anti-E Cadherin antibody [EPR699] (Anti-E Cadherin antibody [EPR699] ab133597) at 1/5000 dilution

  Lane 2: Western blot - Anti-E Cadherin antibody [EPR699] (Anti-E Cadherin antibody [EPR699] ab133597) at 1/2000 dilution

  Lane 2: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/2000 dilution

  Lane 3: Western blot - Anti-E Cadherin antibody [EPR699] (Anti-E Cadherin antibody [EPR699] ab133597)

  Lane 3: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  All lanes: HT-29 (Human colorectal adenocarcinoma epithelial cell). Whole cell lysates at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 97 kDa

  Observed band size: 80 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) secondary antibody at a 1/1000 dilution (green). The nuclear counter stain is DAPI (blue). Counterstained with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker 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.

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  Flow Cytometry (Intracellular) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  Overlay histogram showing A431 (Human epidermoid carcinoma cell line) cells stained with unpurifiedab40772 (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) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/500 dilution for 30 minutes at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/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.
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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.

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

  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)

  All lanes: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/200000 dilution

  All lanes: MCF7 (Human breast adenocarcinoma ) whole cell lysates at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG H&L (HRP) at 1/1000 dilution

  Predicted band size: 97 kDa

  Observed band size: 100 kDa, 120 kDa, 80 kDa, 97 kDa

  Exposure time: 1min

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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.

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  OI-RD Scanning - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody.
  Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  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.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  Tissue Microarrays stained for Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker using ab40772 in immunohistochemical analysis. This table provides a detailed overview of positive (tick mark) and negaive (cross mark) staining per sample type tested. The section was incubated with ab40772 for 30 mins at room temperature followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). The immunostaining was performed on a Leica Biosystems BOND® RX instrument.
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins.

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

  False colour image of Western blot: Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker staining at 1/10000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab40772 was shown to bind specifically to E Cadherin. A band was observed at 105/130 kDa in wild-type Raji cell lysates with no signal observed at this size in CDH1 knockout cell line Human CDH1 (E Cadherin) knockout A-431 cell line ab273747 (knockout cell lysate ab273781). To generate this image, wild-type and CDH1 knockout Raji cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  Lanes 1 - 4: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/10000 dilution

  Lanes 1 - 4: Western blot - Anti-E Cadherin antibody [EP700Y] - Low endotoxin, Azide free (Anti-E Cadherin antibody [EP700Y] - Low endotoxin, Azide free ab201499) at 1/10000 dilution

  Lanes 1 - 4: Western blot - Anti-E Cadherin antibody [EP700Y] - BSA and Azide free (Anti-E Cadherin antibody [EP700Y] - BSA and Azide free ab256580) at 1/10000 dilution

  Lane 1: Wild-type Raji cell lysate at 20 µg

  Lane 2: Western blot - Human CDH1 (E Cadherin) knockout A-431 cell line (Human CDH1 (E Cadherin) knockout A-431 cell line ab273747)

  Lane 2: Western blot - Human CDH1 (E Cadherin) knockout A-431 cell lysate (ab273781)

  Lane 2: CDH1 knockout Raji cell lysate at 20 µg

  Lane 3: MCF7 cell lysate at 20 µg

  Lane 4: MDA-MB-231 cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 105 kDa, 130 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  Immunofluorescence staining of E-Cadherin using ab40772 in wild-type A431 cells (top panel) and CDH1 knockout A431 cells (bottom panel). The cells were fixed with 4% formaldehyde (10 min), 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 ab40772 at 1 µg/mL and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 at 1 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) (shown in red), both at 1/1000. Nuclear DNA was labelled with DAPI (shown in blue).
  
  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a confocal section is shown.

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  Immunocytochemistry/ Immunofluorescence - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772)

  Immunofluorescence staining of E-Cadherin using ab40772 in wild-type A431 cells (top panel) and CDH1 knockout A431 cells (bottom panel). The cells were fixed with 100% methanol (5 min), 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 ab40772 at 0.2 µg/mL and Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 at 1 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) (shown in green) and Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) (shown in red), both at 1/1000. Nuclear DNA was labelled with DAPI (shown in blue).
  
  Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a confocal section is shown.

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

  Fluorescence multiplex immunohistochemical analysis of the human endometrium (Formalin/PFA-fixed paraffin-embedded sections).
  
  Panel A: merged staining of anti-E Cadherin (Anti-E Cadherin antibody [EP700Y] - BSA and Azide free ab256580, red; Opal™690), anti-SLC34A2 (Anti-SLC34A2 antibody [SP322] - BSA and Azide free ab238793, green; Opal™520) and anti-CD10 (Anti-CD10 antibody [EPR22865-73] ab255609, cyan; Opal™570) on human endometrium. Panel B: anti-CD10 stained on stromal cells. Panel C: anti-E Cadherin stained on glandular cells. Panel D: anti-SLC34A2 stained on apical membrane of glandular cells. Opal Polymer HRP Ms + Rb was used as a secondary antibody.
  
  The section was incubated in three rounds of staining: in the order of Anti-E Cadherin antibody [EP700Y] - BSA and Azide free ab256580 at 1/3000 dilution  (0.324 μg/ml) for 30mins, Anti-SLC34A2 antibody [SP322] - BSA and Azide free ab238793 at 1/1000 dilution (2.26 μg/ml) for 10mins and Anti-CD10 antibody [EPR22865-73] ab255609 at 1/1000 dilution (0.615 μg/ml) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system.
  
  The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. Image acquisition was performed with Leica SP8 confocal microscope.
  
  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain.
  
  This data was developed using the same antibody clone in a different buffer formulation (Anti-E Cadherin antibody [EP700Y] - BSA and Azide free ab256580).

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

  Exposure time: 3.25 seconds.
  
  Blocking and diluting buffer: 5% NFDM/TBST.
  
  Full-length E Cadherin has a molecular weight of approximately 125 kDa. Other molecular weights between 80-100 kDa could also be observed depending on cell types or cell conditions.
  
  PMID: 27274359, PMID: 26983597, PMID: 18478055, PMID: 22375065.

  All lanes: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/1000 dilution

  All lanes: MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution

  Predicted band size: 97 kDa

  Observed band size: 80-125 kDa

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

  Western blot: Anti-CDH1 antibody [EP700Y] (ab40772) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab40772 was shown to bind specifically to CDH1. A band was observed at 130, 110, 80, 55, 40 kDa in wild-type A431 cell lysates with no signal observed at this size in CDH1 knockout cell line Human CDH1 (E Cadherin) knockout A-431 cell line ab273747 (knockout cell lysate ab273781). To generate this image, wild-type and CDH1 knockout A431 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-E Cadherin antibody [EP700Y] - Intercellular Junction Marker (ab40772) at 1/1000 dilution

  Lane 1: Wild-type A431 cell lysate at 20 µg

  Lane 2: Western blot - Human CDH1 (E Cadherin) knockout A-431 cell lysate (ab273781)

  Lane 2: Western blot - Human CDH1 (E Cadherin) knockout A-431 cell line (Human CDH1 (E Cadherin) knockout A-431 cell line ab273747)

  Lane 2: CDH1 knockout A431 cell lysate at 20 µg

  Lane 3: Caco-2 cell lysate at 20 µg

  Secondary

  All lanes: Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

  Performed under reducing conditions.

  Predicted band size: 97 kDa

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

  Flow cytometry overlay histogram showing MCF7 positive cells (left) and negative MDA-MB-231 cells (right) stained with ab40772 (red line). The cells were incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction, followed by the antibody ab40772 (1x 10⁶ in 100μl at 5 μg/ml (1/400)) for 30min on ice.

  The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min on ice

  Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

  Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.