Protein lysates were separated on 4–12% SDS-polyacrylamide gels (Life Technologies) and transferred to a nitrocellulose membrane (Life Technologies). Protein detection was performed using an anti-EpCAM monoclonal antibody (R&D Systems, Inc., Minneapolis, MN), anti-β-actin monoclonal antibody (Sigma-Aldrich), anti-E-cadherin monoclonal antibody (Cell Signaling Technology), anti-vimentin monoclonal antibody (Abcam), anti-phospho-Smad2 monoclonal antibody (Cell Signaling), and anti-Cyclin D1 monoclonal antibody (Cell Signaling).
Anti e cadherin monoclonal antibody
Manufactured by Cell Signaling Technology
The Anti-E-cadherin monoclonal antibody is a laboratory reagent used for the detection and analysis of E-cadherin, a cell-cell adhesion protein, in various experimental settings. This antibody can be used to identify and quantify the expression of E-cadherin in cell and tissue samples.
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Lab products found in correlation
Anti vimentin monoclonal antibody, by Cell Signaling Technology (2 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Anti vimentin monoclonal antibody, by Abcam (1 mentions)
Anti β actin monoclonal antibody, by Merck Group (1 mentions)
Goat anti rabbit cy3, by Cell Signaling Technology (1 mentions)
Confocal microscopy, by Olympus (1 mentions)
4 protocols using anti e cadherin monoclonal antibody
1
Western Blot Analysis of Epithelial Markers
2
Immunofluorescence Assay for Epithelial-Mesenchymal Transition
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Huh-7, Bel-7402, SNU387, and SNU449 cells were seeded onto glass slides. At 48 h after transfection with the NAT10 siRNA or Twist siRNA or treatment with remodelin in the presence of doxorubicin or hypoxia, the cells were rinsed with PBS, fixed with 2% paraformaldehyde, permeabilized with 0.1% Triton X-100, blocked for 30 min in 10% BSA, and then incubated with an anti-E-cadherin monoclonal antibody (1 : 200; Cell Signaling Technology) or anti-vimentin monoclonal antibody (1 : 200; Cell Signaling Technology) overnight at 4°C. After three washes in PBS, the slides were incubated with goat anti-rabbit Cy3 as a secondary antibody (1 : 200; Cell Signaling Technology) for 1 h in the dark. After three further washes, the cells were stained with DAPI for 5 min to visualize nuclei and examined by confocal microscopy (Olympus, Tokyo, Japan).
3
Immunohistochemical Analysis of Epithelial-Mesenchymal Transition
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Sections (4 μm) from each block were deparaffinized in xylene and rehydrated in a descending alcohol series. Antigen retrieval was performed by heating in a pressure cooker in 10 mmol/L citrate buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation in 0.3% H2O2 for 15 min, followed by incubation with 5% serum to reduce nonspecific binding. Sections were incubated with an anti-vimentin monoclonal antibody (1 : 1000; Cell Signaling Technology), anti-E-cadherin monoclonal antibody (1 : 1000; Cell Signaling Technology), or anti-Ki-67 monoclonal antibody (1 : 500; Cell Signaling Technology) at 4°C overnight. After washing in phosphate-buffered saline (PBS), slides were incubated with horseradish peroxidase-conjugated rabbit-anti-mouse secondary antibody, developed using 3,3-diaminobenzidine (DAB) chromogen solution and counterstained with Mayer's hematoxylin. Negative controls were performed in parallel by replacing the primary antibody with nonspecific serum.
4
Tissue Microarray Analysis of E-Cadherin in ESCC
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Tissue microarray containing 300 pairs of primary ESCC cases (tumor and corresponding non-tumor tissues) was constructed as described previously [31 (link)]. None of the patients in the study has received follow-up radiation or chemotherapy. IHC was performed using a standard streptavidin-biotin-peroxidase complex method. In brief, a TMA section was deparaffinized and rehydrated. Endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide for 15 minutes. For antigen retrieval, the TMA slide was microwave-treated in 10 mM citrate buffer (pH 6.0) for 10 minutes. The slide was then incubated with anti-E-cadherin monoclonal antibody (Cell Signalling Technology, Danvers, MA). The nucleus was counterstained using Meyer's hematoxylin. E-cadherin immunoreactivity was calculated by adding the scores for the percentage of E-cadherin -positive cells (<5%, 0; 5%–25%, 1; 25%–50%, 2; 50%–75%, 3; >75%, 4) and the intensity of E-cadherin-positive staining (negative, 0; weak, 1; moderate, 2; or strong, 3). Informative results were observed in 237 pairs of ESCC cases, while non-informative samples included lost samples and inappropriate staining.
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