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REACTIVITY SENSITIVITY MW (kDa) Isotype
H Endogenous Mouse IgG1
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Confocal immunofluorescent analysis of HT-29 (left) and HeLa (right) cells using EpCAM (VU1D9) Mouse mAb (Alexa Fluor® 594 Conjugate) (red). Actin filaments were labeled with Alexa Fluor® 488 phalloidin (green). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

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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 1L 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 and store opened vials at 4°C in dark, dilute in 1X PBS for use.
  3. 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.
  4. Image-iT™ FX Signal Enhancer (#11932)
  5. 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.
  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 - 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, 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.
  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. Permeabilize the cells in Permeabilization Buffer for 5 minutes at room temperature.
  2. Rinse three times in 1X PBS for 5 minutes each.
  3. Apply 3–4 drops of Image-iT™ FX Signal Enhancer (#11932) and incubate for 30 minutes at room temperature.
  4. Rinse three times with 1X PBS for 5 minutes each.
  5. Block specimen in Blocking Buffer for 60 minutes.
  6. While blocking, prepare primary antibody by diluting as indicated on datasheet in Antibody Dilution Buffer.
  7. Aspirate blocking solution, apply diluted primary antibody.
  8. Incubate overnight at 4°C.
  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 wavelength. For long-term storage, store slides flat at 4°C protected from light.

posted Februay 2011

protocol id: 6

Product Usage Information

Application Dilutions
Immunofluorescence 1:50

Storage: Supplied in PBS (pH 7.2), less than 0.1% sodium azide and 2 mg/ml BSA. Store at 4°C. Do not aliquot the antibody. Protect from light. Do not freeze.

Specificity / Sensitivity

EpCAM (VU1D9) Mouse mAb (Alexa Fluor® 594 Conjugate) detects endogenous levels of total EpCAM protein.


Species Reactivity: Human

Source / Purification

Monoclonal antibody is produced by immunizing animals with NCI-H69 small cell lung carcinoma cells.

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 594 fluorescent dye and tested in-house for direct immunofluorescent analysis in human cells. The antibody is expected to exhibit the same species cross-reactivity as the unconjugated EpCAM (VU1D9) Mouse mAb #2929.


Epithelial cell adhesion and activating molecule (EpCAM/CD326) is a transmembrane glycoprotein that mediates Ca2+-independent, homophilic adhesions on the basolateral surface of most epithelial cells. EpCAM is not expressed in adult squamous epithelium, but it is highly expressed in adeno and squamous cell carcinomas (1). Research studies identified EpCAM as one of the first tumor-associated antigens, and it has long been a marker of epithelial and tumor tissue. Investigators have shown that EpCAM is highly expressed in cancer cells (reviewed in 2,3).


1.  Went, P.T. et al. (2004) Hum Pathol 35, 122-8.

2.  Baeuerle, P.A. and Gires, O. (2007) Br J Cancer 96, 417-23.

3.  Armstrong, A. and Eck, S.L. (2003) Cancer Biol Ther 2, 320-6.


Entrez-Gene Id 4072
Swiss-Prot Acc. P16422


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.