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REACTIVITY SENSITIVITY MW (kDa) Isotype
H M R Mk Endogenous Rabbit IgG
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Confocal immunofluorescent analysis of HeLa cells using Vimentin (D21H3) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) (blue pseudocolor). Red = Propidium Iodide (fluorescent DNA dye)

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Flow cytometric analysis of HeLa cells, using Vimentin (D21H3) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) (blue) compared to Rabbit (DA1E) mAb IgG XP® Isotype Control (Alexa Fluor® 647 Conjugate) #2985 (red).

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Immunofluorescence (Immunocytochemistry)

A. Solutions and Reagents

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

  2. Allow cells to fix for 15 min at room temperature.
  3. Aspirate fixative, rinse three times in 1X PBS for 5 min 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 min.
  2. While blocking, prepare primary antibody by diluting as indicated on datasheet 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 min each.
  6. Coverslip slides with Prolong® Gold Antifade Reagent (#9071) or Prolong® Gold Antifade Reagent with DAPI (#8961).
  7. 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

protocol id: 182

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Flow Cytometry General Protocol

If using whole blood, please follow the Flow Cytometry Whole Blood Protocol.

A. Solutions and Reagents

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

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. 100% methanol.
  4. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Fixation

  1. Collect cells by centrifugation and aspirate supernatant.
  2. Resuspend cells in 0.5–1 ml 1X PBS. Add formaldehyde to obtain a final concentration of 4%.
  3. Fix for 10 min at 37°C.
  4. Chill tubes on ice for 1 min.
  5. For extracellular staining with antibodies that do not require permeabilization, proceed to immunostaining (Section D) or store cells in PBS with 0.1% sodium azide at 4°C; for intracellular staining, proceed to permeabilization (Section C).

C. Permeabilization

NOTE: This step is critical for many CST antibodies.

  1. Permeabilize cells by adding ice-cold 100% methanol slowly to pre-chilled cells, while gently vortexing, to a final concentration of 90% methanol. Alternatively, remove fix prior to permeabilization by centrifugation and resuspend in 90% methanol as described above.
  2. Incubate 30 min on ice.
  3. Proceed with immunostaining (Section D) or store cells at -20°C in 90% methanol.

D. Immunostaining

NOTE: Account for isotype matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies. Count cells using a hemocytometer or alternative method.

  1. Aliquot 0.5–1 x 106 cells into each assay tube (by volume).
  2. Add 2–3 ml incubation buffer to each tube and wash by centrifugation. Repeat.
  3. Resuspend cells in 100 µl of diluted primary antibody (prepared in incubation buffer at the recommended dilution).
  4. Incubate for 1 hr at room temperature.
  5. Wash by centrifugation in 2–3 ml incubation buffer.
  6. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer; alternatively, for DNA staining, proceed to optional DNA stain (Section E).

E. Optional DNA Dye

  1. Resuspend cells in 0.5 ml of DNA dye (e.g. Propidium Iodide (PI)/RNase Staining Solution #4087).
  2. Incubate for at least 30 min at room temperature.
  3. Analyze cells in DNA staining solution on flow cytometer.

posted July 2009

revised September 2013

Flow Cytometry Whole Blood Protocol

If using cell lines, please follow the Flow Cytometry General Protocol.

A. Solutions and Reagents

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

  1. 20X Phosphate Buffered Saline (PBS): (#9808) To prepare 1 L 1X PBS: add 50 ml 20X PBS to 950 ml dH2O, mix.
  2. 16% Formaldehyde (methanol free).
  3. Triton™ X-100: To prepare 50 ml of 0.1% Triton™ X-100 add 50 μl Triton™ X-100 to 50 ml 1 X PBS and mix well.
  4. 50% methanol.
  5. Incubation Buffer: Dissolve 0.5 g Bovine Serum Albumin (BSA) (#9998) in 100 ml 1X PBS. Store at 4°C.

B. Preparation of Whole Blood (fixation, lysis, and permeabilization) for Immunostaining

  1. Aliquot 100 μl fresh whole blood per assay tube.
  2. OPTIONAL: Place tubes in rack in 37°C water bath for short-term treatments with ligands, inhibitors, drugs, etc.
  3. Add 65 μl of 10% formaldehyde to each tube.
  4. Vortex briefly and let stand for 15 min at room temperature.
  5. Add 1 ml of 0.1% Triton™ X-100 to each tube.
  6. Vortex and let stand for 30 min at room temperature.
  7. Add 1 ml incubation buffer.
  8. Pellet cells by centrifugation and aspirate supernatant.
  9. Repeat steps 7 and 8.
  10. Resuspend cells in ice-cold 50% methanol in PBS (store methanol solution at -20°C until use).
  11. Incubate at least 10 min on ice.
  12. Proceed with staining or store cells at -20°C in 50% methanol.

C. Staining Using Conjugated Primary Antibodies

NOTE: Account for isotype-matched controls for monoclonal antibodies or species matched IgG for polyclonal antibodies.

  1. Add 2–3 ml incubation buffer to each tube and rinse by centrifugation. Repeat.
  2. Add primary antibodies diluted as recommended on datasheet or product webpage in incubation buffer.
  3. Incubate for 1 hr at room temperature.
  4. Wash by centrifugation in 2–3 ml incubation buffer.
  5. Resuspend cells in 0.5 ml PBS and analyze on flow cytometer.

Reference: Chow S, Hedley D, Grom P, Magari R, Jacobberger JW, Shankey TV (2005) Whole blood fixation and permeabilization protocol with red blood cell lysis for flow cytometry of intracellular phosphorylated epitopes in leukocyte subpopulations. Cytometry A 67(1), 4–17.

posted November 2008

revised September 2013

protocol id: 407

Product Usage Information

Application Dilutions
Immunofluorescence 1:400
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.

Specificity / Sensitivity

Vimentin (D21H3) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) detects endogenous levels of total vimentin protein.


Species Reactivity: Human, Mouse, Rat, Monkey

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg45 of human vimentin protein. This antibody was conjugated to Alexa Fluor® 647 under optimal conditions with an F/P ratio of 2-6. The Alexa Fluor® 647 dye is maximally excited by red light (e.g. 633 nm He-Ne laser). Antibody conjugates of the Alexa Fluor® 647 dye produce bright far-red-fluorescence emission, with a peak at 665 nm.

Product Description

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 Vimentin (D21H3) XP® Rabbit mAb #5741.


The cytoskeleton consists of three types of cytosolic fibers: microfilaments (actin filaments), intermediate filaments, and microtubules. Major types of intermediate filaments are distinguished by their cell-specific expression: cytokeratins (epithelial cells), glial fibrillary acidic protein (GFAP) (glial cells), desmin (skeletal, visceral, and certain vascular smooth muscle cells), vimentin (mesenchyme origin), and neurofilaments (neurons). GFAP and vimentin form intermediate filaments in astroglial cells and modulate their motility and shape (1). In particular, vimentin filaments are present at early developmental stages, while GFAP filaments are characteristic of differentiated and mature brain astrocytes. Thus, GFAP is commonly used as a marker for intracranial and intraspinal tumors arising from astrocytes (2). Research studies have shown that vimentin is present in sarcomas, but not carcinomas, and its expression is examined in conjunction with that of other markers to distinguish between the two (3). Vimentin's dynamic structural changes and spatial re-organization in response to extracellular stimuli help to coordinate various signaling pathways (4). Phosphorylation of vimentin at Ser56 in smooth muscle cells regulates the structural arrangement of vimentin filaments in response to serotonin (5,6). Remodeling of vimentin and other intermediate filaments is important during lymphocyte adhesion and migration through the endothelium (7).

During mitosis, CDK1 phosphorylates vimentin at Ser56. This phosphorylation provides a PLK binding site for vimentin-PLK interaction. PLK further phosphorylates vimentin at Ser82, which might serve as memory phosphorylation site and play a regulatory role in vimentin filament disassembly (8,9). Additionally, studies using various soft-tissue sarcoma cells have shown that phosphorylation of vimentin at Ser39 by Akt1 enhances cell migration and survival, suggesting that vimentin could be a potential target for soft-tissue sarcoma targeted therapy (10,11).


1.  Eng, L.F. et al. (2000) Neurochem Res 25, 1439-51.

2.  Goebel, H.H. et al. (1987) Acta Histochem Suppl 34, 81-93.

3.  Leader, M. et al. (1987) Histopathology 11, 63-72.

4.  Helfand, B.T. et al. (2004) J Cell Sci 117, 133-41.

5.  Tang, D.D. et al. (2005) Biochem J 388, 773-83.

6.  Fomina, I.G. et al. (1990) Klin Med (Mosk) 68, 125-7.

7.  Nieminen, M. et al. (2006) Nat Cell Biol 8, 156-62.

8.  Yamaguchi, T. et al. (2005) J Cell Biol 171, 431-6.

9.  Oguri, T. et al. (2006) Genes Cells 11, 531-40.

10.  Zhu, Q.S. et al. (2011) Oncogene 30, 457-70.

11.  Xue, G. and Hemmings, B.A. (2013) J Natl Cancer Inst 105, 393-404.


Entrez-Gene Id 7431
Swiss-Prot Acc. P08670


For Research Use Only. Not For Use In Diagnostic Procedures.
Cell Signaling Technology® is a trademark of Cell Signaling Technology, Inc.
XP® 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.