Formalin-fixed tumors retrieved from the mice were embedded in wax and sections (5 μm) were prepared using standard methods and stained with eosin and hematoxylin. Sections were processed and stained individually for hematoxylin and eosin staining, ER (1:1, Roche), PR (1:1, Roche), HER2 (1:1, Roche), vimentin (1:400, Leica), E-cadherin (1:50, Dako), CK5/6 (1:50, Dako), GATA3 (1:100, Cell Marque), CK20 (1:5000, Biocare), and GCDFP (1:1000, Cell Marque) before observation by microscopy.
E cadherin
Manufactured by Agilent Technologies
Sourced in United States, Denmark, United Kingdom
E-cadherin is a cell adhesion molecule that plays a crucial role in the formation and maintenance of cell-cell junctions. It is a transmembrane protein that facilitates the attachment of cells to one another, contributing to the structural integrity of tissues. E-cadherin is commonly used as a marker in various laboratory applications.
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Lab products found in correlation
Vimentin, by Agilent Technologies (5 mentions)
N cadherin, by Abcam (3 mentions)
Enhanced chemiluminescence western blot detection system, by Cytiva (3 mentions)
Snail, by Biorbyt (3 mentions)
Peroxidase conjugated igg antibodies, by Medical & Biological Laboratories (3 mentions)
Ck5 6, by Agilent Technologies (2 mentions)
Synaptophysin, by Agilent Technologies (2 mentions)
α sma, by Agilent Technologies (2 mentions)
P mek1 2, by Cell Signaling Technology (2 mentions)
Bcl2 4223, by Cell Signaling Technology (2 mentions)
Pfra1, by Cell Signaling Technology (2 mentions)
Melan a m7196, by Agilent Technologies (2 mentions)
P c jun, by Cell Signaling Technology (2 mentions)
Mek1 2, by Cell Signaling Technology (2 mentions)
Vimentin, by BD (2 mentions)
Bond polymer refine kit, by Leica Biosystems (2 mentions)
Bond rx system, by Leica camera (2 mentions)
Bond polymer refine red detection kit, by Leica Biosystems (2 mentions)
Envision detection system, by Agilent Technologies (2 mentions)
Alexa fluor 488, by Thermo Fisher Scientific (2 mentions)
42 protocols using e cadherin
1
Immunohistochemical Analysis of Tumor Markers
2
Immunofluorescence and Immunohistochemistry of Junctional Proteins
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The expression of junctional proteins in cell culture was tested with immunofluorescence using antibodies against connexin 43 (Abcam, Cambridge, UK, 1:100), E-cadherin (Abcam, 1:100), occludin (Novus Biologicals, Littleton, CO, 1:100), and desmoglein (Abcam, 1:10), as described in22 (link). As for paraffin sections, immunohistochemistry was performed using antibodies against connexin 43 (Proteintech, Wuhan, China, 1:200), E-cadherin (Dako, 1:200), occludin (Origene, Rockville, MD, 1:150), and desmoglein (Proteintech, 1:500). The staining was visualized using a Novolink Polymer Detection System (Novocastra Reagents, Wetzlar, Germany). Representative pictures were taken using an Axio Vert. A1 microscope (Carl-Zeiss, Jena, Germany). The intensity of the staining was quantified using ImageJ v1.52a (http://rsb.info.nih.gov/ij/ ). In brief, 20 microphotographs of randomly selected areas of a tumor were taken and analyzed. During the analysis, the area of PMCs was precisely outlined and cells displaying positive color reaction were marked. Then, the area corresponding to positive cells was quantified and expressed as % of the total area of PMCs.
3
Immunofluorescence Analysis of Epithelial-Mesenchymal Transition
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The cells were cultured on coverslips for 12 h, washed with cold PBS three times, and fixed in 4% PFA Fix Solution for 10 min. Subsequently, the cells were blocked with goat serum for 1 h at room temperature and incubated with the anti-β-catenin, E-cadherin, and Vimentin antibody in antibody diluents (Dako) at 4 °C overnight. After being washed, the samples were incubated with the Alex Fluor 488-Donkey anti-rat IgG (H + L; 1:150, Life Technologies, #A21208) and Alex Fluor 594-Donkey anti-rabbit IgG (1:150, Life Technologies, #R37119) for 1 h in the dark at room temperature. The samples were stained with DAPI (4′,6-diamidino-2-phenylindole, Sigma-Aldrich, MO, USA) for 8 min to marker the nucleus and the slices were digitally photographed with a confocal microscope (Leica TCS SP8, Wetzlar, Germany).
4
Immunohistochemical Evaluation of IKK Signaling
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Skin and tumors were fixed in 10% buffered formalin and embedded in paraffin. Sections were stained with H&E and histopathological evaluation was performed by two experimented observers: MJFA, specialized in human pathology and RAGF, a veterinarian expert in animal pathology.
Immunostaining was performed using antibodies against IKKα (NB100-56704) IKKβ (Novus Biologicals, Cambridge UK); IKKα (H00001147-M04) (Abnova, Taiwan); IKKα (sc-7182), P-IKKα/β (Ser 180/Ser 181)-R (sc-23470-R), Maspin, p65 (Santa Cruz Biotechnology, Inc. Heidelberg, Germany); CD31, E- Cadherin, Integrin-α6 (BD Bioscience, NJ, USA); p52 (Abcam, Cambridge, UK). Sections were incubated with a biotinylated secondary antibody, and then with streptavidin conjugated to horseradish peroxidase (DAKO A/S, Glostrp, Denmark). Antibody localization was determined using 3,3-diaminobenzidine (DAB) (Vector Laboratories; Burlingame, CA, USA).
A pressure cooker with DAKO target retrieval solution ph9.0 (DAKO) was employed for Maspin, mouse IKKα, human IKKα, P-IKKα/β, IKKβ and E- Cadherin detection. Staining with p52, p65, Integrin-α6 and CD31 antibodies was performed in cryosections of tumors.
Immunostaining was performed using antibodies against IKKα (NB100-56704) IKKβ (Novus Biologicals, Cambridge UK); IKKα (H00001147-M04) (Abnova, Taiwan); IKKα (sc-7182), P-IKKα/β (Ser 180/Ser 181)-R (sc-23470-R), Maspin, p65 (Santa Cruz Biotechnology, Inc. Heidelberg, Germany); CD31, E- Cadherin, Integrin-α6 (BD Bioscience, NJ, USA); p52 (Abcam, Cambridge, UK). Sections were incubated with a biotinylated secondary antibody, and then with streptavidin conjugated to horseradish peroxidase (DAKO A/S, Glostrp, Denmark). Antibody localization was determined using 3,3-diaminobenzidine (DAB) (Vector Laboratories; Burlingame, CA, USA).
A pressure cooker with DAKO target retrieval solution ph9.0 (DAKO) was employed for Maspin, mouse IKKα, human IKKα, P-IKKα/β, IKKβ and E- Cadherin detection. Staining with p52, p65, Integrin-α6 and CD31 antibodies was performed in cryosections of tumors.
5
Immunocytochemical Analysis of Cell Adhesion
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In order to investigate immunocytochemical reactions, control and stimulated cells were formalin-fixed and paraffin embedded before obtaining 4–5 mm-thick sections from each cell block. All immunocytochemical staining was performed automatically (Bondt—maX, Menarini, Florence, Italy) with primary antibodies: E-cadherin (DakoCytomation, Glostrup, Denmark) (1:200), N-cadherin (DakoCytomation) (1:100), Twist1 (Novocastra, Newcastle-upon-Tyne, UK)(1:600). Sections were then lightly counterstained with hematoxylin. E-cadherin and N-cadherin immunoreactions were defined as immunoreactions detectable in the membrane while Twist was defined as a positive nuclear staining. At least three independent sets of experiments were performed in each experimental condition.
6
Comprehensive Protein Expression Analysis in EMT
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Akt2 (5B5), p-AKT (S473; 736E11), p44/42 MAPK (Erk1/2; 137F5), p-p44/42 MAPK (T202/Y204, D13.14.4E), p-Smad2 (S465/467; 138D4), Smad4, SNAI2, ZEB1 (all Cell Signaling Technology); AGR2 (K-31, in-house); AGR2 (1C3, Abnova); vimentin (V9, Dako); E-cadherin (NCH38, Dako; HECD1, Abcam; Cell Signaling); N-cadherin (3B9, Invitrogen); β-actin (C4, Santa Cruz Biotech); Alexa Fluor 488 goat anti-mouse IgG, Alexa Fluor 532 goat anti-rabbit IgG (both Abcam); α-tubulin (AA13, Sigma); HRP-conjugated swine anti-rabbit and HRP-conjugated rabbit anti-mouse (both Dako).
7
Quantifying Notch2 Activation in Monolayers
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Monolayer cell cultures at 80% confluence were analyzed by western blotting as previously described (8 (link)), using primary antibodies against human activated NOTCH2 (ab72803; Abcam), E-cadherin (Dako) or β-actin (Sigma).
8
Immunohistochemical Profiling of Liver Tumors
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Antibodies directed against HepPar1, glypican3, EpCAM, cytokeratin 19 (K19), αSMA, (clone 1A4), vimentin (Clone V9) and E-cadherin were obtained from Dakocytomation, Glostrup, Denmark. Anti-γSMA antibodies (Clone E184) were obtained from Epitomics. Anti-Snail, anti-Twist and anti-V5 antibodies were sourced from Abcam. Anti-p38 antibody came from SantaCruz Biotechnology, and anti-calcitonin antibody was from Diagnostic Biosystems.
9
Immunohistochemical Profiling of Tissue Samples
10
Immunohistochemical analysis of TLR7, E-cadherin, and Vimentin
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Human tumor samples were fixed in neutral-buffered 10% formalin solution and paraffin embedded. TLR7 staining and quantification was performed as previously described 11 using TLR7-specific polyclonal antibody (ENZO Lifesciences) at 10 mg/mL.
The expression of E-cadherin (Dako M3613, 1/100 dilution) and vimentin (Cells Signaling 5741, 1/100) by tumor cells was performed as follows. Serial 5-μm tissue sections were deparaffinized, rehydrated, and pretreated in 10 mM citrate buffer, pH 6, for antigen retrieval. Sections were incubated with hydrogen peroxide for 15 minutes, then blocked in 5% human serum for 30 minutes at room temperature. The slides were then incubated with a primary antibody (diluted in Dako real solution, antibody diluent) anti-E-cadherin (Dako M3613, 1/100 dilution) for one hour at room temperature or anti-vimentin (Cells Signaling 5741, 1/100) overnight. Slides were then incubated for 30 minutes at room temperature with the secondary antibody (anti-rabbit coupled to biotin, 1/200, JIR 715–066-150), followed by 30 minutes incubation with streptavidin-HRP (Dako, 1/300). After each incubation, the slides were washed 5 minutes with 1x TBS+ 0.04% Tween. Revelation was performed with the DAB kit (Dako, K3468) and stopped by placing slides in 1X TBS and distillated water. Counter coloration was performed with hematoxylin.
The expression of E-cadherin (Dako M3613, 1/100 dilution) and vimentin (Cells Signaling 5741, 1/100) by tumor cells was performed as follows. Serial 5-μm tissue sections were deparaffinized, rehydrated, and pretreated in 10 mM citrate buffer, pH 6, for antigen retrieval. Sections were incubated with hydrogen peroxide for 15 minutes, then blocked in 5% human serum for 30 minutes at room temperature. The slides were then incubated with a primary antibody (diluted in Dako real solution, antibody diluent) anti-E-cadherin (Dako M3613, 1/100 dilution) for one hour at room temperature or anti-vimentin (Cells Signaling 5741, 1/100) overnight. Slides were then incubated for 30 minutes at room temperature with the secondary antibody (anti-rabbit coupled to biotin, 1/200, JIR 715–066-150), followed by 30 minutes incubation with streptavidin-HRP (Dako, 1/300). After each incubation, the slides were washed 5 minutes with 1x TBS+ 0.04% Tween. Revelation was performed with the DAB kit (Dako, K3468) and stopped by placing slides in 1X TBS and distillated water. Counter coloration was performed with hematoxylin.
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