The cultured cells were lysed with Pierce RIPA Buffer (Thermo Scientific, Waltham, MA, USA) with Halt protease inhibitor Cocktail (Thermo Scientific). Lysates mixed with sample buffer were electrophoretically separated and transferred onto membranes. Membranes were blocked with 5% skim milk, followed by incubations with anti-human E-cadherin antibody (24E10; Cell Signaling Technology, Danvers, MA, USA), anti-human N-cadherin antibody (#610920; BD Biosciences, San Jose, CA, USA), anti-human vimentin antibody (D21H3; Cell Signaling Technology), anti-human Smad2/3 (#07-408; Millipore, Billerica, MA, USA), anti-human phospho-Smad2 (Ser465/467) (#AB3849; Millipore), anti-human phospho-Smad3 (Ser423/425) (#07-1389; Millipore), and anti-human β-actin antibody (#4963; Cell Signaling Technology). After washing with TBS-0.05% Tween, membranes were incubated with HRP-conjugated anti-mouse or anti-rabbit IgG. After washing with TBS-0.05% Tween, membranes were incubated with ECL Prime Western Blotting Detection Reagent (GE Healthcare, Little Chalfont, UK). Signals were detected and analysed using a Luminescent Image Analyzer LAS-4000 (Fuji Film, Tokyo, Japan). Signals were detected and analysed using a Luminescent Image Analyzer LAS-4000 (Fuji Film).
Anti human e cadherin antibody
Manufactured by Cell Signaling Technology
Sourced in United States, Germany
The Anti-human E-cadherin antibody is a laboratory tool used to detect and analyze the E-cadherin protein in human samples. E-cadherin is a cell-cell adhesion molecule that plays a crucial role in maintaining the integrity of epithelial tissues. This antibody can be utilized in various research applications, such as Western blotting, immunohistochemistry, and immunocytochemistry, to study the expression and localization of E-cadherin in different biological systems.
Automatically generated - may contain errors
Lab products found in correlation
Las 4000 luminescent image analyzer, by Fujifilm (1 mentions)
Pierce ripa buffer, by Thermo Fisher Scientific (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Halt protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Anti human β actin antibody, by Cell Signaling Technology (1 mentions)
Las 4000, by Cytiva (1 mentions)
Biorevo bz 9000 fluorescent microscope, by Keyence (1 mentions)
Falcon cultureslides, by Corning (1 mentions)
3 protocols using anti human e cadherin antibody
1
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
2
Immunohistochemical Analysis of Protein Expression
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Immunohistochemistry (IHC) analysis was performed on 4-μm-thick sections of paraffin-embedded tumor tissue specimens. A monoclonal anti-human EIF5A2 antibody, anti-human E-cadherin antibody, anti-human vimentin antibody, and anti-human c-myc antibody were purchased from Cell Signaling Technology and were used for IHC according to the manufacturer’s instructions. Briefly, 4-μm-thick tissue sections cut from formalin-fixed, paraffin-embedded blocks were deparaffinized with xylene and rehydrated with ethanol. Endogenous peroxidase activity was blocked with 3% H2O2 in methanol. After antigen retrieval, the sections were incubated with the primary antibodies overnight at 4 °C. The sections were washed with PBS three times, and then incubated with a secondary antibody. After washing, a color reaction was induced by the IHC Streptavidin-Biotin Complex method and 3,3ʹ-diaminobenzidine staining. The results of the IHC assay were independently reviewed by two senior pathologists who were blinded to the outcome of the study. The staining intensity of the target proteins was scored with reference to a previous study.17 (link)
3
Immunocytochemistry of Epithelial and Mesenchymal Markers
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The cells were cultivated for 72 h, with subsequent fixation with 4% paraformaldehyde in chamber slides (Falcon® Culture Slides; Corning, NY, USA and Sarstedt, Nümbrecht, Germany). The cells were permeabilized with 0.1% Triton-X and sequentially treated with goat serum, primary monoclonal mouse anti-human CK 7 antibody (DAKO Corporation, Clone OV-TL 12/30, 1:200 dilution), primary monoclonal rabbit anti-human E-cadherin antibody (Cell Signaling Technology, Frankfurt/Main, Germany, #3195, 1:200 dilution), primary monoclonal mouse anti-human vimentin antibody (Abcam, Cambridge, UK, V1-RE/1, ab3974,1:200 dilution), anti-Mouse Alexa Fluor® 594 secondary antibody (Abcam, #150116, 1:500 dilution), and anti-Rabbit Alexa Fluor® 488 secondary antibody (Abcam, #150077, 1:500 dilution). Appropriate negative controls were performed. Coverslips were mounted with Fluoromount-G medium including DAPI (Southern Biotech, Huntsville, AL, USA). The cells were visualized using a BZ-9000 (BIOREVO) fluorescent microscope (Keyence, Osaka, Japan) with a 20× PlanFluor EL NA 0.45 Ph1 objective lens under 0.285 s of exposure time for CK7 and 0.2 s of exposure time for E-cadherin and vimentin.
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