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EpCAM (VU1D9) Mouse mAb (Alexa Fluor® 647 Conjugate) #5447
Gallery: EpCAM (VU1D9) Mouse mAb (Alexa Fluor® 647 Conjugate) #5447
A. Solutions and Reagents
NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade 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.
- 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.
- 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 diluted in 1X PBS.
NOTE: Formaldehyde is toxic, use only in a fume hood.
- Allow cells to fix for 15 min at room temperature.
- Aspirate fixative, rinse three times in 1X PBS for 5 min 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.
- Block specimen in Blocking Buffer for 60 min.
- 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 min each.
- Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
- For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 4°C protected from light.
posted November 2006
revised November 2013
A. Solutions and Reagents
- 1X Phosphate Buffered Saline (PBS): Dissolve 8 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4 and 0.24 g KH2PO4 in 800 mL distilled water (dH2O). Adjust the pH to 7.4 with HCl and the volume to 1 liter. Store at room temperature.
- Formaldehyde (methanol free).
- Incubation Buffer: Dissolve 0.5 g bovine serum albumin (BSA) in 100mL 1X PBS. Store at 4°C.
- Collect cells by centrifugation and aspirate supernatant.
- Resuspend cells briefly in 0.5-1 ml PBS. Add formaldehyde to a final concentration of 2-4% formaldehyde.
- Fix for 10 minutes at 37°C.
- Add 5 ml PBS and rinse by centrifugation.
- Resuspend cells in 5 ml PBS.
- Proceed with staining or store cells at +4°C in PBS with 0.1% sodium azide.
NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.
- Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
- Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
- Resuspend cells in 100 µl incubation buffer per assay tube.
- Block in Incubation Buffer for 10 min at room temperature.
- Add fluorochrome-conjugated primary antibody at the appropriate dilution to the assay tubes (see individual antibody datasheet or product webpage for the appropriate dilution).
- Incubate for 1 hr at room temperature.
- Wash by centrifugation in 2–3 ml incubation buffer.
- Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).
posted January 2009
EpCAM (VU1D9) Mouse mAb (Alexa Fluor® 647 Conjugate) detects endogenous levels of total EpCAM protein.Species Reactivity: Human
Monoclonal antibody is produced by immunizing animals with NCI-H69 small cell lung carcinoma cells.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 647 fluorescent dye and tested in-house for direct flow cytometry and 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).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. 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.