Mouse monoclonal anti n cadherin

Manufactured by BD

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Mouse monoclonal anti-N-cadherin is a laboratory reagent used to detect the presence of N-cadherin, a cell adhesion protein, in biological samples. It is a mouse-derived monoclonal antibody that specifically binds to N-cadherin.

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N-cadherin (256 citations) Anti-N-cadherin (132 citations) Mouse anti-N-cadherin (19 citations) Mouse anti (2 citations)

6 protocols using mouse monoclonal anti n cadherin

Immunofluorescence Characterization of Endothelial Cell-Tumor Cell Interactions

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Fixation was carried out by adding an equal volume of 2× fixative (PBS with 4% paraformaldehyde and 0.4% glutaraldehyde) to EC monolayers in EGM-2 MV medium 15 min, 1 hr, 3 hrs, and 6 hrs after addition of UM cells. After 15 min at 37°C, cells were permeabilized with 0.1% Triton X-100 in PBS for 5 min, washed with PBS, and blocked with 2% fish gelatin (Sigma-Aldrich) in PBS. Primary and secondary antibodies were diluted in 2% fish gelatin in PBS. Primary antibodies included mouse monoclonal anti-cortactin (Millipore, Billerica, MA), mouse monoclonal anti-E-cadherin (BD Biosciences, Franklin Lakes, NJ), mouse monoclonal anti-N-cadherin (BD Biosciences), mouse monoclonal anti-VCAM (R&D Systems, Minneapolis, MN), rabbit polyclonal anti-S100 (DakoCytomation, Denmark), and rabbit polyclonal anti-VE-cadherin (Cell Signaling, Danvers, MA). F-actin was visualized using Alexa-fluor-conjugated phalloidin (Life Technologies). Secondary antibodies were Alexa-fluor conjugates (Life Technologies), and the mounting agent was ProLong Gold (Life Technologies). Cells were imaged with an inverted microscope (Olympus IX72) using a 40× objective.

Onken M.D., Li J, & Cooper J.A. (2014). Uveal Melanoma Cells Utilize a Novel Route for Transendothelial Migration. PLoS ONE, 9(12), e115472.

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Immunostaining of Cell Junctions

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Cell pairs were fixed with 4% paraformaldehyde (Electron Microscopy Services) in phosphate-buffered saline (PBS) for 30 minutes, or with ice-cold 100% methanol (Sigma) for 5min. After PBS washes, cells were permeabilized for 5 minutes using 0.1% Triton X-100 in PBS. Cells were then blocked for 1 hour at room temperature in 5% normal goat serum (Life Technologies) before addition of primary antibodies. Mouse monoclonal anti-N-cadherin (BD Biosciences), chick anti-GFP (Abcam), rabbit anti-Cx43 (Sigma), mouse monoclonal anti-EB1 (BD Biosciences), mouse monoclonal anti-αTubulin (Sigma), and Alexa Fluor 555 Phalloidin (Life Technologies) were incubated for 1 hour with 5% normal goat serum in PBS at a dilution of 1:500 each. Following three washes with PBS, cell pairs were incubated for 1 hour with goat Alexa Fluor secondary antibodies (Life Technologies) and TO-PRO-3 nuclear counterstain (Life Technologies). ProLong gold (with DAPI) mounting reagent (Life Technologies) was added prior to confocal imaging.

Zhang S.S., Hong S., Kléber A.G., Lee L.P, & Shaw R.M. (2014). A micropatterning approach for imaging Cx43 dynamic trafficking to cell-cell borders. FEBS letters, 588(8), 1439-1445.

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Immunofluorescence Staining of Cardiac Tissues

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Samples were fixed in 2% paraformaldehyde for 15 min at RT. Autoflourescence quenching was performed by incubating samples in 0.1 M NH₄Cl (pH 8.0) for 10 min at RT, and then in a fresh NH₄Cl solution for an additional 5 min. This was followed by antigen retreival via incubation in pre-warmed 10 mM trisodium citrate dihydrate (pH 6.0) for 15 minutes. Samples were then permeablized in PBS solution containing Triton X-100 (0.2% v/v), sucrose (10% w/v), and magnesium chloride (0.6% w/v) for 10 min and blocked in 1% (w/v) bovine serum albumin. Tissues were incubated with mouse monoclonal anti-N-cadherin (1:200; BD Bioscience 610921) overnight at 4 °C, rabbit monoclonal anti-connexin43 (1:1000; Millipore Sigma AB1728) antibodies overnight at 4 °C, mouse monoclonal anti-α-actinin (1:500; Abcam ab9465) for 1 hour at RT, mouse monoclonal anti-cardiac troponin T (1:500; ThermoFisher MA5-12960) for 1 hour at RT, mouse monoclonal or rabbit monoclonal anti-Ki67 (1:1000; Sigma-Aldrich PIMA514520) for 1 hour at RT, followed by either goat anti-mouse Alex Fluor 647 (1:1000; Life Technologies A21236) or goat anti-rabbit Alexa Fluor 647 secondary antibodies, (1:1000; Life Technologies A21245) and DAPI for 1 hour at RT.

DePalma S.J., Davidson C.D., Stis A.E., Helms A.S, & Baker B.M. (2021). Microenvironmental determinants of organized iPSC-cardiomyocyte tissues on synthetic fibrous matrices. Biomaterials science, 9(1), 93-107.

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Immunostaining of Cell-Cell Adhesion Proteins

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A549 and MDCK cells were cultured in glass slides. After confluency, cells were fixed with 3.7% formaldehyde and permeabilized with 0.2% Triton X-100. Cells were incubated with mouse monoclonal anti-E-cadherin (1:50, BD Biosciences, San Jose, CA, USA), mouse monoclonal anti-β-catenin (1:200, BD Biosciences, San Jose, CA, USA), mouse monoclonal anti-N-cadherin (1:50, BD Biosciences, San Jose, CA, USA) overnight at 4 °C, and they were washed three times with PBS for 15 min each. Subsequently, the cells were incubated with alexa fluor 488 goat anti-mouse IgG secondary antibodies (1:100 for E- and N-cadherin and 1:400 for β-catenin, Thermo Fisher Scientific, Waltham, MA, USA) and with 2 µg/ml of Hoechst 33342 dye for nuclear staining for 60 min at room temperature. After incubation, the samples were rinsed 3 times with PBS for 15 min each. Finally, the samples were examined under an inverted fluorescence microscope (Olympus, Tokyo, JP).

Wu Y.H., Lee Y.H., Shih H.Y., Chen S.H., Cheng Y.C, & Tsun-Yee Chiu D. (2018). Glucose-6-phosphate dehydrogenase is indispensable in embryonic development by modulation of epithelial-mesenchymal transition via the NOX/Smad3/miR-200b axis. Cell Death & Disease, 9(1), 10.

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Protein Expression Analysis of Differentiated ES Cells

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ES cells were treated with 1 μM RA for 4 days. The cells were lysed in RIPA buffer containing protease and phosphatase inhibitors, and protein concentration was measured by BioRad-DCC protein assay. The lysates were diluted 1:1 with BioRad 2X loading buffer, and equal protein was loaded on 4-12% Tris glycine-polyacrylamide (Invitrogen) gels. Separated protein was electrotransferred to nitrocellulose membrane (Pall, Pensacola, FL), and immunoblotted with primary antibodies. Antibodies used were: mouse monoclonal anti-lamin A/C (1:1000, Active Motif, #39288); rabbit polyclonal anti-emerin (1:1000, ProteinTech, #10351-1-AP); mouse monoclonal anti-actin (1:5000, BD-Transduction, #612656); rabbit polyclonal anti-ERK2/1 (1:1000, Cell Signaling, #9102); mouse monoclonal anti-Dab2 (1:2000, BD-Transduction, #610464); rabbit polyclonal anti-Gata4 (1:1000, Santa Cruz, sc9053); mouse monoclonal anti-Oct3/4 (1:1000, Santa Cruz, sc5279); rabbit anti-Nanog (1:1000, Abcam, ab80892); mouse monoclonal anti-N-cadherin (1:1000, BD-Transduction, #610920). HRP-conjugated secondary antibodies were from BioRad and used at 1:5000 dilution. SuperSignal West Dura Extended Duration substrate (Thermo Scientific, #34076) was used to visualize the immunoblots.

Smith E.R., Meng Y., Moore R., Tse J.D., Xu A.G, & Xu X.X. (2017). Nuclear envelope structural proteins facilitate nuclear shape changes accompanying embryonic differentiation and fidelity of gene expression. BMC Cell Biology, 18, 8.

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Western Blot Analysis of EMT Markers

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Cells were lysed in RIPA buffer (150 mM NaCl, 50 mM Tris base, 5 mM EDTA, 1% NP-40, 0.25% deoxycholate, pH 7.4) with protease and phosphatase inhibitors (Roche, Complete Mini). Protein concentrations were measured by the BCA protein assay (23227; Thermo Fisher Scientific, Waltham, MA, USA). Equal amounts (30–50 μg) of the protein were electrophoresed by SDS-PAGE, transferred to NC membranes, and incubated with the following primary antibodies: anti-β-actin antibody (Cell Signaling Technology, Danvers, MA, USA), rabbit polyclonal anti-E-cadherin (A01589; GenScript, Edison, NJ, USA), mouse monoclonal anti-N-cadherin (BD Transduction, San Jose, CA, USA), rabbit polyclonal anti-vimentin (A01189; GenScript), rabbit monoclonal anti-β-catenin (8480; Cell Signaling Technology), rabbit monoclonal anti-Snail (3879; Cell Signaling Technology), and rabbit monoclonal anti-Twist (ab50581; Abcam, Cambridge, MA, USA). The primary antibody incubation was followed by incubation with an HRP-conjugated secondary antibody. The bound antibodies were detected using enhanced chemiluminescence reagent (32109; Thermo Fisher Scientific).

Zeng J., Du T., Song Y., Gao Y., Li F., Wu R., Chen Y., Li W., Zhou H., Yang Y, & Pei Z. (2017). Knockdown of Long Noncoding RNA CCAT2 Inhibits Cellular Proliferation, Invasion, and Epithelial–Mesenchymal Transition in Glioma Cells. Oncology Research, 25(6), 913-921.

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