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Vimentin (D21H3) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #9855
Gallery: Vimentin (D21H3) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) #9855
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
protocol id: 182
Vimentin (D21H3) XP® Rabbit mAb (Alexa Fluor® 555 Conjugate) detects endogenous levels of total vimentin protein.Species Reactivity: Human, Mouse, Rat, Monkey
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Arg45 of human vimentin protein.
This Cell Signaling Technology antibody is conjugated to Alexa Fluor® 555 fluorescent dye and tested in-house for 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).
For Research Use Only. Not For Use In Diagnostic Procedures. Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc. XP is a registered 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.