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7771
PathScan® EMT Duplex IF Kit
Antibody Cocktail Kit
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  2. PathScan® EMT Duplex IF Kit

PathScan® EMT Duplex IF Kit #7771

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Immunofluorescence Image 1: PathScan® EMT Duplex IF Kit
Confocal immunofluorescent analysis of paraffin-embedded human kidney using PathScan® EMT Duplex IF Kit. Green = E-cadherin, red = vimentin, and blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
Immunofluorescence Image 1: PathScan® EMT Duplex IF Kit
Confocal immunofluorescent analysis of cocultured MCF7 and MDA-MB-231 cells using PathScan® EMT Duplex IF Kit. Green = E-cadherin, red = vimentin, and blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).
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Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

Product Description

The PathScan® EMT Duplex IF Kit offers a novel method to simultaneously monitor cells of epithelial or mesenchymal origin, as well as those undergoing an epithelial-mesenchymal transition (EMT) using manual immunofluorescence microscopy or automated imaging and laser scanning high content platforms. This kit contains a cocktail of two high quality primary antibodies targeted against vimentin and E-cadherin, as well as a detection cocktail utilizing the Alexa Fluor® series of fluorescent dyes. Antibody and dye pairings have been pre-optimized and each kit contains enough reagents for 100 assays (based on a working volume of 100 μl/test).

Protocol

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Immunofluorescence (Paraffin)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. Xylene.
  2. Ethanol, anhydrous denatured, histological grade, 100% and 95%.
  3. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  4. Antigen Unmasking Citrate: 10 mM Sodium Citrate Buffer: To prepare 1 L add 2.94 g sodium citrate trisodium salt dihydrate (C6H5Na3O7•2H2O) to 1 L dH2O. Adjust pH to 6.0.
  5. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100):
    To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100
  6. 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.
  7. Prolong® Gold AntiFade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).

B. Specimen Preparation - Paraffin Sections (IF-P)

NOTE: Do not allow slides to dry at any time during this procedure.

Deparaffinization/Rehydration:

  1. Incubate sections in three washes of xylene for 5 minutes each.
  2. Incubate sections in two washes of 100% ethanol for 10 minutes each.
  3. Incubate sections in two washes of 95% ethanol for 10 minutes each.
  4. Rinse sections twice in dH2O for 5 minutes each.

Antigen Unmasking:

NOTE: Consult protocol on product webpage for specific recommendation for the unmasking solution.

  1. For Citrate: Bring slides to a boil in 10 mM sodium citrate buffer pH 6.0 then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench top for 30 minutes.

C. Immunostaining

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.

  1. Rinse three times in 1X PBS for 5 minutes each.
  2. Block specimen in Blocking Buffer for 60 minutes.
  3. While blocking, prepare primary cocktail by diluting as indicated on protocol on product webpage in Antibody Dilution Buffer.
  4. Aspirate blocking solution, apply diluted primary cocktail.
  5. Incubate overnight at 4°C.
  6. Rinse three times in 1X PBS for 5 minutes each.
  7. Prepare detection cocktail by diluting as indicated on protocol on product webpage in Antibody Dilution Buffer.
  8. Incubate 1–2 hours at room temperature in the dark.
  9. Rinse three times in 1X PBS for 5 minutes each.
  10. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  11. For best results examine specimens immediately using appropriate excitation wavelengths. For long-term storage, store slides at 4°C protected from light.

posted July 2010

revised August 2011

Protocol Id: 445

Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalently purified water.

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix. Adjust pH to 8.0.
  2. Formaldehyde, 16%, methanol free, Polysciences, Inc. (cat# 18814), use fresh, store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. Blocking Buffer (1X PBS / 5% normal goat serum (#5425) / 0.3% Triton™ X-100):
    To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix. While stirring, add 30 µl Triton™ X-100
  4. 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.
  5. 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.

  1. Aspirate liquid, and then cover cells to a depth of 2–3 mm with 4% formaldehyde diluted in 1X PBS.
    NOTE: Formaldehyde is toxic, use only in fume hood.
  2. Allow cells to fix for 15 minutes at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 minutes each.
  4. Proceed with immunostaining (Section C).

C. Immunostaining

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.

  1. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary cocktail by diluting as indicated on product webpage in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary cocktail.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Prepare detection cocktail by diluting as indicated on product webpage in Antibody Dilution Buffer.
  7. Incubate 1–2 hours at room temperature in the dark.
  8. Rinse three times in 1X PBS for 5 minutes each.
  9. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), or Prolong® Gold Antifade Reagent with DAPI (#8961).
  10. For best results examine specimens immediately using appropriate excitation wavelengths. For long-term storage, store slides at 4°C protected from light.

posted July 2010

revised August 2011

Protocol Id: 424

Specificity / Sensitivity

Vimentin mAb detects endogenous levels of total vimentin protein. E-cadherin mAb detects endogenous levels of total E-cadherin protein and does not cross react with related family members such as N-cadherin.

Species Reactivity:

Human

Source / Purification

Monoclonal antibodies were produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg45 of human vimentin protein or residues surrounding Pro780 of human E-cadherin protein.

Background

Epithelial-mesenchymal transition (EMT) refers to a biological process in which cells undergo a series of biochemical changes that induce a morphological transformation from an epithelial, polarized, adhesive state to an irregular, elongated, mesenchymal phenotype that enables migratory capacity (1,2). EMTs are classified into three subtypes: those involved in implantation, embryogenesis, and organ development; those associated with inflammation and fibrosis; and those involved in invasion and metastasis (1). Molecular changes that are associated with cells during this transformation include the loss of E-cadherin and gain of vimentin expression, hallmark epithelial and mesenchymal markers, respectively (3-5). Numerous studies have established that EMT is an essential step in cancer metastasis (5-7). E-cadherin is regarded as an active suppressor of invasion and tumorigenesis (8). In response to extracellular stimuli, vimentin coordinates various signaling pathways to induce spatial reorganization and structural changes (9), reminiscent of the EMT phenotype observed in motile cells involved in invasion and metastasis (6).
  1. Kalluri, R. and Weinberg, R.A. (2009) J Clin Invest 119, 1420-8.
  2. Lee, J.M. et al. (2006) J Cell Biol 172, 973-81.
  3. Yan, W. et al. (2010) J Biol Chem 285, 14042-51.
  4. Sethi, S. et al. (2011) Transl Oncol 4, 222-6.
  5. Kim, J.H. et al. (2007) J Korean Med Sci 22, 898-904.
  6. Emadi Baygi, M. et al. (2010) Cell Biol Toxicol 26, 553-67.
  7. Sethi, S. et al. (2010) Am J Transl Res 3, 90-9.
  8. Wheelock, M.J. and Johnson, K.R. (2003) Annu Rev Cell Dev Biol 19, 207-35.
  9. Helfand, B.T. et al. (2004) J Cell Sci 117, 133-41.

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Products are labeled with For Research Use Only or a similar labeling statement and have not been approved, cleared, or licensed by the FDA or other regulatory foreign or domestic entity, for any purpose. Customer shall not use any Product for any diagnostic or therapeutic purpose, or otherwise in any manner that conflicts with its labeling statement. Products sold or licensed by CST are provided for Customer as the end-user and solely for research and development uses. Any use of Product for diagnostic, prophylactic or therapeutic purposes, or any purchase of Product for resale (alone or as a component) or other commercial purpose, requires a separate license from CST. Customer shall (a) not sell, license, loan, donate or otherwise transfer or make available any Product to any third party, whether alone or in combination with other materials, or use the Products to manufacture any commercial products, (b) not copy, modify, reverse engineer, decompile, disassemble or otherwise attempt to discover the underlying structure or technology of the Products, or use the Products for the purpose of developing any products or services that would compete with CST's products or services, (c) not alter or remove from the Products any trademarks, trade names, logos, patent or copyright notices or markings, (d) use the Products solely in accordance with CST's Product Terms of Sale and any applicable documentation, and (e) comply with any license, terms of service or similar agreement with respect to any third party products or services used by Customer in connection with the Products.

For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
PathScan is a trademark of Cell Signaling Technology, Inc.
DRAQ5 is a registered trademark of Biostatus Limited.
The transfer of the secondary cocktail contained in this kit is contingent on the buyer using the purchased product solely in research conducted by the buyer (whether the buyer is an academic or for-profit entity), for immunocytochemistry, high content screening (HCS) analysis, or flow cytometry applications. The sale of this product is expressly conditioned on the buyer not using the product or its components (1) in manufacturing; (2) to provide a service, information, or data to an unaffiliated third party for payment; (3) for therapeutic, diagnostic or prophylactic purposes; (4) resale, whether or not such product or its components are resold for use in research; or for any other commercial purpose. For information on purchasing a license to this product for purposes other than research, contact Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008 USA or [email protected] Alexa Fluor® is a registered trademark of Molecular Probes, Inc.