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E-Cadherin (24E10) Rabbit mAb (Alexa Fluor® 594 Conjugate) #7687
Gallery: E-Cadherin (24E10) Rabbit mAb (Alexa Fluor® 594 Conjugate) #7687
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.
- 20X Phosphate Buffered Saline (PBS): (9808) To prepare 1L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
- Formaldehyde: 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh and store opened vials at 4°C in dark, dilute in 1X PBS for use.
- Permeabilization Buffer (1X PBS/0.2% Triton X-100):
To prepare 25 ml, add 2.5 ml 10X PBS, and 22.5 ml dH2O and mix well. While stirring, add 50 µl Triton X-100.
- Image-iT™ FX Signal Enhancer (#11932)
- Blocking Buffer (1X PBS / 5% normal serum / 0.3% Triton™ X-100): To prepare 10 ml, add 0.5 ml normal serum from the same species as the secondary antibody (e.g., Normal Goat Serum (#5425)) and 0.5 mL 20X PBS to 9.0 mL dH2O, mix well. While stirring, add 30 µl Triton™ X-100.
- Antibody Dilution Buffer (1X PBS / 1% BSA / 0.3% Triton X-100): To prepare 10 ml, add 30 µl Triton™ X-100 to 10 ml 1X PBS. Mix well then add 0.1 g BSA (9998), mix.
- Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
B. Specimen Preparation - Cultured Cell Lines (IF-IC)
NOTE: Cells should be grown, treated, fixed, and stained directly in multi-well plates, chamber slides, or on coverslips.
- Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% formaldehyde in 1X PBS.
NOTE: Formaldehyde is toxic, use only in fume hood.
- Allow cells to fix for 15 minutes at room temperature.
- Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
- Proceed with Immunostaining (Section C).
NOTE: All subsequent incubations should be carried out at room temperature unless otherwise noted in a humid light-tight box or covered dish/plate to prevent drying and fluorochrome fading.
- Permeabilize the cells in Permeabilization Buffer for 5 minutes at room temperature.
- Rinse three times in 1X PBS for 5 minutes each.
- Apply 3–4 drops of Image-iT™ FX Signal Enhancer (#11932) and incubate for 30 minutes at room temperature.
- Rinse three times with 1X PBS for 5 minutes each.
- Block specimen in Blocking Buffer for 60 minutes.
- While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
- Aspirate blocking solution, apply diluted primary antibody.
- Incubate overnight at 4°C.
- Rinse three times in 1X PBS for 5 minutes each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
- For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.
posted Februay 2011
protocol id: 6
E-Cadherin (24E10) Rabbit mAb (Alexa Fluor® 594 Conjugate) recognizes endogenous levels of total E-cadherin protein. The antibody does not cross-react with related family members, such as N-cadherin.Species Reactivity: Human, Mouse Species predicted to react based on 100% sequence homology: Bovine, Dog, Pig
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to the sequence surrounding Pro780 of human E-cadherin protein.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 594 fluorescent dye and tested in-house for immunofluorescent analysis in human cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated E-Cadherin (24E10) Rabbit mAb #3195.
Cadherins are a superfamily of transmembrane glycoproteins that contain cadherin repeats of approximately 100 residues in their extracellular domain. Cadherins mediate calcium-dependent cell-cell adhesion and play critical roles in normal tissue development (1). The classic cadherin subfamily includes N-, P-, R-, B-, and E-cadherins, as well as about ten other members that are found in adherens junctions, a cellular structure near the apical surface of polarized epithelial cells. The cytoplasmic domain of classical cadherins interacts with β-catenin, γ-catenin (also called plakoglobin), and p120 catenin. β-catenin and γ-catenin associate with α-catenin, which links the cadherin-catenin complex to the actin cytoskeleton (1,2). While β- and γ-catenin play structural roles in the junctional complex, p120 regulates cadherin adhesive activity and trafficking (1-4). Investigators consider E-cadherin an active suppressor of invasion and growth of many epithelial cancers (1-3). Research studies indicate that cancer cells have up-regulated N-cadherin in addition to loss of E-cadherin. This change in cadherin expression is called the "cadherin switch". N-cadherin cooperates with the FGF receptor, leading to overexpression of MMP-9 and cellular invasion (3). Research studies have shown that in endothelial cells, VE-cadherin signaling, expression, and localization correlate with vascular permeability and tumor angiogenesis (5,6). Investigators have also demonstrated that expression of P-cadherin, which is normally present in epithelial cells, is also altered in ovarian and other human cancers (7,8).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. DRAQ5 is a registered trademark of Biostatus Limited. The Alexa Fluor dye antibody conjugates in this product are sold under license from Life Technologies Corporation for research use only, except for use in combination with DNA microarrays. The Alexa Fluor® dyes (except for Alexa Fluor® 430 dye) are covered by pending and issued patents. Alexa Fluor® is a registered trademark of Molecular Probes, Inc. U.S. Patent No. 5,675,063.