MSCs were seeded onto Type I collagen (190 μg/ml; Nitta Gelatin NA Inc., Morrisville, NC, USA) coated cover glasses. The cells were washed with ice-cold PBS (pH 7.0) containing Ca2+ and Mg2+ and fixed with 4% formaldehyde (Sigma-Aldrich) for 10 min on ice. Permeabilization was conducted with 0.2% Triton X-100 (Sigma-Aldrich) in PBS, and 5% non-fat milk in 0.1% Triton X-100 in PBS was used for the blocking. The cells were incubated for 90 min at 25 °C with primary antibodies against ZO-1 (Thermo Fisher Scientific), α-catenin (BD Biosciences), N-cadherin (Thermo Fisher Scientific), N-cadherin (Takara, Shiga, Japan), or Myc-tag (Cell Signaling Technology). Nuclei were stained using 4,6-diamidino-2-phenylindole (DAPI; Thermo Fisher Scientific) for 10 min, and actin was detected using Alexa-Fluor-633-tagged phalloidin (Thermo Fisher Scientific). The covers were mounted on glass slides with Fluoromount-G (SouthernBiotech, Birmingham, AL, USA). Image acquisition was performed by Zeiss LSM 700 confocal microscope (Carl Zeiss, Oberkochen, Germany) and processed by Zeiss Zen (Blue edition) software. If necessary, consecutive stitch sequences were processed into a single image using Fiji 2.14 (NIH, Bethesda, MD, USA).
N cadherin
Manufactured by Takara Bio
Sourced in Japan
N-cadherin is a cell-surface protein that mediates calcium-dependent cell-cell adhesion. It is a member of the cadherin family of proteins and is expressed in various cell types, including neural and epithelial cells.
Automatically generated - may contain errors
Lab products found in correlation
Alexa fluor 488 and 568, by Thermo Fisher Scientific (2 mentions)
Recombinant human tgf β1, by R&D Systems (2 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (2 mentions)
Paxillin, by BD (2 mentions)
E cadherin, by BD (2 mentions)
Secondary horse radish peroxidase conjugated antibodies against mouse and rabbit, by Jackson ImmunoResearch (2 mentions)
Triton x 100, by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
N cadherin, by Thermo Fisher Scientific (1 mentions)
α catenin, by BD (1 mentions)
Lsm 700 confocal microscope, by Zeiss (1 mentions)
Myc tag, by Cell Signaling Technology (1 mentions)
Fluoromount g, by Southern Biotech (1 mentions)
α tubulin, by Merck Group (1 mentions)
Gapdh, by Abcam (1 mentions)
Vimentin, by Novus Biologicals (1 mentions)
Alexa fluor 568 phalloidin, by Thermo Fisher Scientific (1 mentions)
Fibronectin1, by Merck Group (1 mentions)
E cadherin, by Zymo Research (1 mentions)
Tb green premix ex taq 2 kit, by Takara Bio (1 mentions)
5 protocols using n cadherin
1
Microscopic Analysis of Cell-Cell Junctions
2
Epithelial Mesenchymal Transition Protein Analysis
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E-cadherin (BD Transduction Labs, 610182; used for immunoblotting), E-cadherin (Zymed, 13-1900; used for immunofluorescence stainings), N-cadherin (Takara, M142), Zona Occludens-1 (Zymed, 617300), Paxillin (BD, 610052), Fibronectin1 (Sigma-Aldrich, F3648), Vimentin (Novus Biological, NB300–223), α-Tubulin (Sigma, T-9026), GAPDH (Abcam, ab9485), Alexa-Fluor 488 and 568 (Molecular Probes), secondary horse radish peroxidase (HRP)-conjugated antibodies against mouse and rabbit (Jackson ImmunoResearch), Phalloidin Alexa-Fluor 568 (Molecular Probes, A12380), 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich, D9542), recombinant human TGFβ1 (R&D Systems, 240-B).
3
Epithelial-Mesenchymal Transition Evaluation
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UA was purchased from Sigma-Aldrich (Merck KGaA). RPMI-1640 medium, PBS and penicillin-streptomycin were purchased from Hyclone (GE Healthcare Life Sciences). Fetal bovine serum (FBS) and trypsin-EDTA were obtained from Gibco (Thermo Fisher Scientific. Inc.). 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) was purchased from Beijing Solarbio Science & Technology Co., Ltd. miR-200a/b/c and U6 primers were synthesized by Takara Biotechnology Co., Ltd. RNAiso for small RNA kit, Mir-X™ miRNA First-Strand Synthesis kit and TB Green™ Premix Ex Taq II kit were purchased from Takara Biotechnology Co., Ltd. N-cadherin (cat. no. ab18203) and E-cadherin (cat. no. ab1416) antibodies were purchased from Abcam. TGF-β1 (cat. no. 3711), phosphorylated (p-) Smad2/3 (cat. no. 8828), Smad2/3 (cat. no. 8685), p-FAK (cat. no. 3284), FAK (cat. no. 71433), ZEB1 (cat. no. 3396) and horseradish peroxidase (HRP)-conjugated secondary antibodies (anti-rabbit IgG; cat. no. 7074; and anti-mouse IgG; cat. no. 7076) were purchased from Cell Signaling Technology, Inc. The β-actin antibody (cat. no. 66009-1-Ig) was purchased from ProteinTech Group, Inc. The Transwell chambers were obtained from Corning Life Sciences and the BD BioCoat Matrigel Invasion Chamber was purchased from BD Bioscience. All the other chemicals, unless otherwise stated, were obtained from Sigma-Aldrich (Merck KGaA).
4
Epithelial-Mesenchymal Transition Protein Analysis
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E-cadherin (BD Transduction Labs, 610182), N-cadherin (Takara, M142), ZO-1 (Zymed, 617300), paxillin (BD, 610052), fibronectin (Sigma-Aldrich, F3648), vimentin (Sigma-Aldrich, V2258), GAPDH (Sigma-Aldrich, G8795), Zeb1 (Cell Signaling, 3396 and Santa Cruz Biotechnology, sc-25388), Alexa-Fluor 488 and 568 (Molecular Probes), secondary horse radish peroxidase (HRP)-conjugated antibodies against mouse and rabbit (Jackson ImmunoResearch), recombinant human TGFβ1 (R&D Systems, 240-B), 4′,6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich) and phalloidin Alexa-Fluor 568 (Molecular Probes, A12380).
5
Immunohistochemical Evaluation of Epithelial-Mesenchymal Transition Markers
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Immunostaining was performed on at least one representative paraffin section using routine laboratory standard protocols. The antibodies used on paraffin-embedded tissues included EMA (Dako, Glostrup, Denmark), CK7 (Dako), CK20 (Dako), E-cadherin (Nichirei, Tokyo, Japan), N-cadherin (TaKaRa, Otsu, Japan), Vimentin (Dako), Fibronectin (Abcam, Cambridge, UK), Snail (Abcam) and CD138 (Dako). The stained tumor tissues were evaluated blindly with respect to clinical patient data. Staining was assessed using a semiquantitative scoring system (0, 1+, 2+, and 3+). Immunohistochemical staining was evaluated as follows: 0, no staining of tumor cells; 1+, faint staining in less than 10% of tumor cells; 2+, weak or moderate staining in more than 10% of tumor cells; and 3+, strong staining in more than 10% of tumor cells. Staining intensity of 0 or 1+ was considered negative, while 2+ or 3+ staining was considered positive. Negative controls were incubated without the primary antibody.
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