Proteins from cells or tissues were extracted with RIPA lysis buffer (Pierce, MA, USA). Equal amount of protein was separated by SDS denatured polyacrylamide gel (SDS-PAGE) and transferred onto PVDF membrane (Millipore, Billerica, MA, USA). Then, the membrane was blocked with 5% (w/v) nonfat milk, incubated with specific primary antibodies at 4°C overnight and HRP-labelled secondary antibodies at 37°C for 1 h. The enhanced chemiluminescence system (ImageQuant LAS4000mini, GE, Japan) was used to visualize the protein band signals. The antibodies were as follows: rabbit anti-β-actin antibody (1 : 5000; Abclonal, Wuhan, China), rabbit anti-Vimentin antibody (1 : 800; Bioworld, USA), rabbit anti-E-cadherin antibody (1 : 300; Santa Cruz, USA), rabbit anti-N-Cadherin antibody (1 : 300; Cell Signaling, USA), rabbit anti-MMP2 antibody (1 : 300; Cell Signaling, USA), rabbit anti-MMP9 antibody (1 : 300; Cell Signaling, USA), rabbit anti-GNG7 antibody (1 : 500; ABclonal, USA), and Goat anti-Rabbit IgG Secondary Antibody (1 : 2000; Invitrogen, USA). The densitometry readings of each band were obtained using ImageJ program, and the fold change of indicated proteins was determined by normalizing to β-actin expression.
Rabbit anti e cadherin antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Rabbit anti-E-cadherin antibody is a primary antibody that specifically binds to the E-cadherin protein, a cell-cell adhesion molecule. It can be used for the detection and analysis of E-cadherin expression in various research applications.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Rabbit anti vimentin antibody, by Bioworld Technology (1 mentions)
Rabbit anti n cadherin antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti mmp9 antibody, by Cell Signaling Technology (1 mentions)
Rabbit anti β actin antibody, by ABclonal (1 mentions)
Goat anti rabbit igg secondary antibody, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
In cell analyzer 6000 system, by GE Healthcare (1 mentions)
Alexa fluor 488 conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Ix71 fluorescence microscope, by Olympus (1 mentions)
Anti cd31 rabbit polyclonal antibody, by Abcam (1 mentions)
Rabbit anti iba 1 polyclonal antibody, by Fujifilm (1 mentions)
Dapi fluoromount g, by Southern Biotech (1 mentions)
Bz x800, by Keyence (1 mentions)
Complete mini protease inhibitor, by Roche (1 mentions)
Cell culture lysis reagent, by Promega (1 mentions)
Pierce ecl plus reagent, by Thermo Fisher Scientific (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Las 3000, by Fujifilm (1 mentions)
6 protocols using rabbit anti e cadherin antibody
1
Western Blot Analysis of Cell Proteins
2
Immunofluorescence Analysis of Keratinocyte Differentiation
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Immunofluorescence analysis was performed as previously described [18 (link)] with slight modifications. In brief, HaCaT cells were seeded at a density of 5 × 103 cells per well on a 24-well plate and cultured in calcium-free medium. After pretreatment with 3% SPE for 1 h, cells were treated with 2 mM CaCl2 to induce keratinocyte differentiation. After 72 h, cells were fixed in 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and then blocked in 5% bovine serum albumin. Next, cells were incubated with rabbit anti-E-cadherin antibody (1:200) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) overnight at 4 °C. After washing with PBS, cells were incubated with Alexa Fluor 488-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) for 1 h. After re-washing with PBS, fluorescent and bright-field images were obtained using an IN Cell Analyzer 6000 system (GE Healthcare UK Ltd., Little Chalfont, Buckinghamshire, UK).
3
Immunofluorescence Microscopy of E-cadherin and Vimentin
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For the in vitro component, the cells were washed with cold PBS three times and fixed in 4% paraformaldehyde at room temperature for 30 min. The cells were then blocked with 3% bovine serum albumin for 1 h at room temperature. For E-cadherin and vimentin staining, the cells were incubated overnight at 4 °C with rabbit anti E-cadherin antibody and rabbit anti-vimentin antibody (1:200, Santa Cruz, Dallas, TX, USA). On the next day, the cells were washed three times with cold PBS and incubated with Alexa Fluor 488-conjugated goat-anti-rabbit IgG (1:200, Jackson ImmunoResearch, West Grove, PA, USA) at 37 °C for 1 h. Subsequently, the cells were washed three times with cold PBS and incubated with 4′,6-diamidino-2-phenylindole (DAPI) for nuclear staining at room temperature for 10 min. Images were visualized using an Olympus IX71 fluorescence microscope (Olympus, Tokyo, Japan). For the in vivo component, the paraffin-embedded lung sections were deparaffinized and rehydrated before staining. The mean intensity of fluorescence obtained from the different groups was analyzed using Image J software.
4
Immunohistochemical Analysis of Cotton Rat Tissues
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The sections were deparaffinized, heated with 0.01 M sodium citrate buffer (pH6.0; LSI Medience, Tokyo, Japan) for 40 min at 95°C, and incubated with 2% bovine serum albumin for 30 min, followed by rabbit anti-Iba1 polyclonal antibody (1:3000, Fujifilm Wako), rabbit anti-E-cadherin antibody (1:100, Santa Cruz, TX, USA), and rabbit anti-CD31 polyclonal antibody (1:100, Abcam, Cambridge, UK) overnight at approximately 4°C. Next, the sections were treated with Alexa Fluor 594 donkey anti-rabbit IgG antibodies (1:1000; Life Technologies, Carlsbad, CA, USA) for 30 min at approximately 25°C, and coverslipped with DAPI Fluoromount-G (SouthernBiotech, AL, USA). The sections were observed using an all-in-one fluorescence microscope (BZ-X800, Keyence) (cotton rat, n=4).
5
E-cadherin Immunoblotting in MCF10A Cells
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MCF10A and MCF10A CDH1-/- cells were grown for 72 h to 90% confluency in T25 flasks and lysed using cell culture lysis reagent (Promega) containing cOmplete mini protease inhibitor (Roche). BCA assays (Thermo) were performed to equalize total protein loaded. Proteins were separated on 10% SDS-PAGE gel for 2 h, followed by blot transfer at 100 V for 1 h. Immunoblotting was performed using rabbit anti-E-cadherin antibody (Santa Cruz, SC7870) at 1:200 dilution overnight, or rabbit anti-α-actin primary antibody (Sigma) at 1:1,500 dilution overnight followed by anti-rabbit HRP-linked secondary antibody (Santa Cruz) at 1:5,000 dilution for 1 h. Chemiluminescence was performed using Pierce ECLplus reagent (Thermo) and imaged using LAS-3000 (Fujifilm).
6
Immunostaining Protocol for LCMV Nucleoprotein and CA IX
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Primary antibodies: We have used undiluted hybridoma medium of mouse monoclonal antibody M87, specific for nucleoprotein of LCMV strain MX [15 (link), 77 (link)], undiluted hybridoma medium of mouse monoclonal antibody M75 (specific for the PG domain of the CA IX protein) [77 (link)] and VII/20 antibody (internalizing monoclonal antibody, specific for CA domain of the CA IX protein) [51 (link)]. Purified mouse anti-human HIF-1α antibody diluted 1:500 (BD Transduction, San Jose, CA, USA); rabbit anti HIF-2α antibody diluted 1:500 (Novus Biologicals, Littleton, CO, USA); mouse anti-enolase antibody in concentration 2 μg/ml (Abcam, Cambridge, UK); rabbit anti-lactate dehydrogenase-A (LDH-A) antibody diluted 1:1000 (Abcam), rabbit anti-β-catenin antibody diluted 1:500 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit anti-E-cadherin antibody diluted 1:500 (Santa Cruz Biotechnology) and mouse anti β-actin diluted 1:5000 (Cell Signalling Technology, Danvers, MA, USA) were also used.
Secondary antibodies: polyclonal goat anti-mouse immunoglobulins conjugated with horseradish peroxidase, diluted 1:5000 (Dako, Glostrup, Denmark) and polyclonal swine anti-rabbit immunoglobulins conjugated with horseradish peroxidase, diluted 1:5000 (Dako). Donkey anti-mouse IgG (H + L) secondary antibody conjugated with Alexa Fluor 488 (Termo Fisher Scientific, Paisley, Scotland).
Secondary antibodies: polyclonal goat anti-mouse immunoglobulins conjugated with horseradish peroxidase, diluted 1:5000 (Dako, Glostrup, Denmark) and polyclonal swine anti-rabbit immunoglobulins conjugated with horseradish peroxidase, diluted 1:5000 (Dako). Donkey anti-mouse IgG (H + L) secondary antibody conjugated with Alexa Fluor 488 (Termo Fisher Scientific, Paisley, Scotland).
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