Immunohistochemical (IHC) staining and evaluation were performed as previously described 9 (link). Antibodies against MEDAG (Biorbyt, orb380371), E-cadherin (E-cad; Cell Signaling Technology, 14472), N-cadherin (N-cad; Cell Signaling Technology, 13116) and Ki67 (Santa Cruz, sc-23900) were used.
N cadherin n cad
Manufactured by Cell Signaling Technology
Sourced in China, United States
N-cadherin (N-cad) is a calcium-dependent cell adhesion molecule that plays a role in cell-cell interactions. It is involved in the formation and maintenance of adherens junctions between cells.
Automatically generated - may contain errors
Lab products found in correlation
E cadherin e cad, by Cell Signaling Technology (2 mentions)
Gapdh, by Abcam (2 mentions)
E cad, by Cell Signaling Technology (1 mentions)
E cadherin, by Cell Signaling Technology (1 mentions)
Heat shock protein 70, by Abcam (1 mentions)
Bicinchoninic acid protein determination kit, by Thermo Fisher Scientific (1 mentions)
C casp3, by Cell Signaling Technology (1 mentions)
C parp, by Cell Signaling Technology (1 mentions)
Vimentin vim, by Cell Signaling Technology (1 mentions)
β actin actb, by Cell Signaling Technology (1 mentions)
Mmp 2, by Cell Signaling Technology (1 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (1 mentions)
Bcl 2, by Affinity Biosciences (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Roche (1 mentions)
Fluorescent microscope, by Olympus (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ecl detection system, by Vazyme (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
Ripa lysate, by Solarbio (1 mentions)
Polyvinylidene difluoride membrane, by GE Healthcare (1 mentions)
5 protocols using n cadherin n cad
1
Immunohistochemical Evaluation of EMT Markers
2
Protein Extraction and Western Blot Analysis from Diverse Biological Samples
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Proteins were extracted from MC38 cells, liver tissues, CRLM tissues, CRLM-adjacent tissues, and exosomes by a lysis buffer (50 mmol/L Tris, 1% NP40, 0.25% deoxycholic acid sodium salt, 150 mmol/L NaCl, 1 mmol/L EGTA), and protein concentrations were determined using a bicinchoninic acid protein determination kit (Thermo Scientific, Waltham, MA) according to the manufacturer’s instructions. Western blot analyses were performed with antibodies against SMPD3 (Santa Cruz Biotechnology), MMP2 (Cell Signaling Technology), cCASP3 (Cell Signaling Technology), cPARP (Cell Signaling Technology), BCL-2 (Affinity, Changzhou, Jiangsu, China), BAX (Cell Signaling Technology), cytochrome C (CYC) (Abcam), heat shock protein 90 (Abcam), heat shock protein 70 (Abcam), CD63 (Abcam), CD9 (Proteintech, Guangzhou, China), N-cadherin (NCAD) (Cell Signaling Technology), vimentin (VIM) (Cell Signaling Technology), E-cadherin (ECAD) (Cell Signaling Technology), CANTB (Cell Signaling Technology), CD31(Abcam), GAPDH (Abcam), Histong3 H3 (Abcam), and β-Actin (ACTB) (Cell Signaling Technology). ACTB was used as a loading control. The information on these primary antibodies is listed in Table 2 .
3
Immunofluorescence Analysis of U251 Cells
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U251 cells were grown on 4-well sterile glass slides for 24 h in 6-well plates to reach 80-90% confluence at 37°C. Following serum starvation for 12 h, cells were washed with PBS three times, immobilized with 4% paraformaldehyde at room temperature for 20 min, permeabilized with 0.1% Triton X-100 at room temperature for 10 min and blocked with 3% bovine serum albumin (BSA; Sigma-Aldrich; Merck KGaA) at 37°C for 90 min. Then, cells were incubated with primary antibodies recognizing Ki67 (cat. no. 11882S; 1:200; Cell Signaling Technology, Inc.), E-cadherin (E-cad; cat. no. 3195T; 1:200; Cell Signaling Technology, Inc.) and N-cadherin (N-cad; cat. no. sc-8424; 1:200; Santa Cruz Biotechnology, Inc.) at 4°C overnight followed by probing with the DyLight™ 488-conjugated secondary antibody (cat. no. ab96899; 1:250; Abcam) at room temperature for 2 h. DAPI (Roche Diagnostics) was used to stain the nuclei in the dark at room temperature for 5 min. Images were taken under an Olympus fluorescent microscope (Olympus Corporation; magnification, ×200).
4
Evaluating EMT Markers in GC Tissues
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Proteins were extracted from GC tissues using RIPA lysate (SolarBio Life Science, Beijing, China). Tissue protein lysis products were electrolyzed using 12% SDS/PAGE (Shanghai EpiZyme Biotechnology, Shanghai, China). Thereafter, they were transferred to a polyvinylidene–fluoride membrane (Millipore, Billerica, MA, USA). Immunoblots were visualized by an ECL detection system (Vazyme Biotech Co., Ltd.). Antibodies against GADPH were used as controls. Antibodies against GADPH, N‐cadherin (N‐cad), and vimentin were obtained from Cell Signaling Technology, Danvers, MA, USA. All three antibodies were diluted at 1 : 1000.
5
Evaluating EMT-related Protein Levels by Western Blotting
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Western blotting (WB) was performed to determine the expression levels of proteins associated with epithelial-mesenchymal transition (EMT). The total intracellular proteins were extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China), and the protein concentration was determined using bicinchoninic acid (Pierce; Thermo Fisher Scientific, Inc.). A total of 50 µg protein was separated on 10-12% SDS-PAGE (Beyotime Institute of Biotechnology) and transferred to polyvinylidene difluoride membranes (GE Healthcare) and then blocked with 5% non-fat milk at room temperature for 2 h. Primary antibodies: E-cadherin (E-cad; cat. no. 14472; Cell Signaling Technology, Inc.), N-cadherin (N-cad; cat. no. 14215; Cell Signaling Technology, Inc.), Snail (cat. no. ab53519; Abcam), SP1 (cat. no. ab13370; Abcam) and GAPDH (cat. no. ab8245; Abcam) at a dilution of 1:1,000 at 4°C were used to incubate the membranes overnight. GAPDH served as internal reference. Next, a horseradish peroxidase-labeled secondary antibody (dilution, 1:1,000; cat. no. ab6728; Abcam) was added to incubate the membranes for another hour at room temperature. An enhanced chemiluminescence kit (Thermo Fisher Scientific, Inc.) was used for color development.
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