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64022
CD15/SSEA1 (MC480) Mouse mAb (Alexa Fluor® 488 Conjugate)
Antibody Conjugates
Monoclonal Antibody

CD15/SSEA1 (MC480) Mouse mAb (Alexa Fluor® 488 Conjugate) #64022

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  2. F
Immunofluorescence Image 1: CD15/SSEA1 (MC480) Mouse mAb (Alexa Fluor® 488 Conjugate)
Confocal immunofluorescent analysis of mouse embryonic stem cells (left, positive) and NIH/3T3 cells (right, negative) using CD15/SSEA1 (MC480) Mouse mAb (Alexa Fluor® 488 Conjugate) (green), DyLight️ 554 Phalloidin #13054 (red), and DAPI #4083 (blue).
Flow Cytometry Image 1: CD15/SSEA1 (MC480) Mouse mAb (Alexa Fluor® 488 Conjugate)
Flow cytometric analysis of unpermeabilized NIH/3T3 cells (blue) and unpermeabilized F9 cells (green) using CD15/SSEA1 (MC480) Mouse mAb (Alexa Fluor® 488 Conjugate) (solid lines) or concentration-matched IgM Isotype (Alexa Fluor® 488 Conjugate) (dashed lines).
To Purchase # 64022
Cat. # Size Qty. Price
64022S
100 µl  (50 tests) $ 336

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Supporting Data

REACTIVITY M
SENSITIVITY Endogenous
MW (kDa)
Source/Isotype Mouse IgM

Application Key:

  • WB-Western Blot
  • IP-Immunoprecipitation
  • IHC-Immunohistochemistry
  • ChIP-Chromatin Immunoprecipitation
  • IF-Immunofluorescence
  • F-Flow Cytometry
  • E-P-ELISA-Peptide

Species Cross-Reactivity Key:

  • H-Human
  • M-Mouse
  • R-Rat
  • Hm-Hamster
  • Mk-Monkey
  • Vir-Virus
  • Mi-Mink
  • C-Chicken
  • Dm-D. melanogaster
  • X-Xenopus
  • Z-Zebrafish
  • B-Bovine
  • Dg-Dog
  • Pg-Pig
  • Sc-S. cerevisiae
  • Ce-C. elegans
  • Hr-Horse
  • All-All Species Expected

Product Description

This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 488 fluorescent dye and tested in-house for direct flow cytometric and immunofluorescent analysis in mouse cells. This antibody is expected to exhibit the same species cross-reactivity as the unconjugated CD15/SSEA1 (MC480) Mouse mAb #4744.

Product Usage Information

Application Dilution
Immunofluorescence (Immunocytochemistry) 1:50
Flow Cytometry 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.

Protocol

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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. Blocking Buffer (1X PBS / 5% normal goat serum (#5425)): To prepare 10 ml: add 0.5 ml normal goat serum and 0.5 ml 20X PBS to 9.0 ml dH2O, mix.
  4. Antibody Dilution Buffer (1X PBS / 1% BSA): Add 0.1 g BSA (9998) to 10 ml 1X PBS and mix well.
  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, 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. Block specimen in Blocking Buffer for 60 minutes.
  2. While blocking, prepare primary antibody by diluting as indicated on product webpage in Antibody Dilution Buffer.
  3. Aspirate blocking solution, apply diluted primary antibody.
  4. Incubate overnight at 4°C.
  5. Rinse three times in 1X PBS for 5 minutes each.
  6. Coverslip slides with Prolong® Gold Antifade Reagent (#9071), Prolong® Gold AntiFade Reagent with DAPI (#8961).
  7. For best results, examine specimens immediately using appropriate excitation wavelength. For long-term storage, store slides flat at 4°C protected from light.

posted December 2010

Protocol Id: 223

Flow Cytometry, Extracellular Epitope Protocol for Direct Conjugates

A. Solutions and Reagents

NOTE: Prepare solutions with reverse osmosis deionized (RODI) or equivalent grade water.

  1. 1X Phosphate Buffered Saline (PBS): To prepare 1 L 1X PBS: add 100 ml 10X PBS (#12528) to 900 ml water, mix.
  2. 4% Formaldehyde, Methanol-free (#47746), Optional
  3. Antibody Dilution Buffer: Purchase ready-to-use Flow Cytometry Antibody Dilution Buffer (#13616), or prepare a 0.5% BSA PBS buffer by dissolving 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

NOTE: When including fluorescent cellular dyes in your experiment (including viability dyes, DNA dyes, etc.), please refer to the dye product page for the recommended protocol. Visit www.cellsignal.com for a full listing of cellular dyes validated for use in flow cytometry.

NOTE: Optimal centrifugation conditions will vary depending upon cell type and reagent volume. Generally, 150-300g for 1-5 minutes will be sufficient to pellet the cells.

B. Fixation

NOTE: If live cell staining is desired, proceed to Section C.

  1. Pellet cells by centrifugation and remove supernatant.
  2. Resuspend cells in approximately 100 µl 4% formaldehyde per 1 million cells. Mix well to dissociate pellet and prevent cross-linking of individual cells.
  3. Fix for 15 min at room temperature (20-25°C).
  4. Wash by centrifugation with excess 1X PBS. Discard supernatant in appropriate waste container. Resuspend cells in 1X PBS.

C. Immunostaining

NOTE: Count cells using a hemocytometer or alternative method.

  1. Aliquot desired number of cells into tubes or wells. (Generally, 5x105 to 1x106 cells per assay.)
  2. Pellet cells by centrifugation and remove supernatant.
  3. Resuspend cells in 100 µl of diluted antibody conjugate, prepared in Antibody Dilution Buffer at the recommended dilution.
  4. Incubate for 30-60 minutes at room temperature (20-25°C).
  5. Wash by centrifugation in Antibody Dilution Buffer. Discard supernatant. Repeat.
  6. Resuspend cells in 200-500 µl of 1X PBS and analyze on flow cytometer.

posted January 2009

revised June 2020

Protocol Id: 180

Specificity / Sensitivity

CD15/SSEA1 (MC480) Mouse mAb (Alexa Fluor® 488 Conjugate) detects endogenous levels of SSEA1 antigen.

Species Reactivity:

Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with F9 teratoma cells (X-irradiated).

Background

SSEA-1 antibody detects a lactoseries oligosaccharide antigen that is expressed on the surface of mouse embryonal carcinoma and embryonic stem cells (1). This antigen is also found on early mouse embryos and both mouse and human germ cells, but is absent on human embryonic stem cells and human embryonic carcinoma cells. Expression of SSEA1 in these human cell types increases upon differentiation, while on the mouse cell types differentiation leads to decreased expression (2).
  1. Solter, D. and Knowles, B.B. (1978) Proc Natl Acad Sci USA 75, 5565-9.
  2. Fenderson, B.A. et al. (1987) Dev Biol 122, 21-34.

Pathways & Proteins

Explore pathways + proteins related to this product.

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For Research Use Only. Not For Use In Diagnostic Procedures.
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