Vimentin Antibody, FITC
Description
Definition and Core Characteristics
Vimentin Antibody, FITC consists of monoclonal or polyclonal antibodies chemically linked to FITC, enabling fluorescence-based detection of vimentin (57 kDa), a structural protein in mesenchymal cells and tumors of mesenchymal origin (e.g., sarcomas, lymphomas) . Key attributes include:
Epitope Specificity
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Clone VIM 3B4: Targets the α-helical rod domain (coil 2) near amino acid 353. Weakly reacts with murine vimentin due to residue differences .
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Clone RV202: Binds the C-terminal region (amino acids 409–466) .
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Clone V9: Recognizes surface vimentin on activated macrophages and apoptotic cells .
Cross-Reactivity and Limitations
| Species | Reactivity | Key Studies |
|---|---|---|
| Human | High | |
| Bovine | High | |
| Chicken | High | |
| Mouse | Low/None | Weak (VIM 3B4) ; None (V9) |
Key Use Cases
Therapeutic Research Findings
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Angiogenesis Inhibition: Anti-vimentin antibodies disrupt tumor vascularization by blocking extracellular vimentin’s mimicry of VEGF signaling, enhancing immune cell infiltration .
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Immune Checkpoint Role: Extracellular vimentin upregulates PD-L1 on endothelial cells, promoting immune evasion. Antibody targeting reverses this effect .
Technical Considerations
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Storage: Stable at 4°C (short-term) or -20°C (long-term); avoid freeze-thaw cycles .
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Controls: Include vimentin-positive (e.g., fibroblasts) and negative (epithelial cells) samples .
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Limitations: Murine models require validation due to variable cross-reactivity .
Emerging Research Directions
Product Specs
Q&A
What is the primary function of Vimentin in cellular biology?
Vimentin is a cytoskeletal intermediate filament protein predominantly expressed in mesenchymal cells. It plays a crucial role in maintaining cellular integrity, providing structural support, and facilitating intracellular transport. Vimentin is involved in processes such as cell migration, adhesion, proliferation, and epithelial-to-mesenchymal transition (EMT), which are critical during wound healing and cancer metastasis . Additionally, vimentin has been implicated in regulating autophagy and cell size through its interaction with the mTORC1 signaling pathway .
What are the recommended applications for Vimentin Antibody, FITC?
The Vimentin Antibody conjugated with FITC is widely used in immunocytochemistry (ICC), immunohistochemistry (IHC), flow cytometry, and Western blotting. These applications allow researchers to detect vimentin expression in various cell types and tissues under different experimental conditions . For example:
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Immunocytochemistry (ICC): Enables visualization of vimentin localization within cells.
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Flow Cytometry: Facilitates quantitative analysis of vimentin expression on cell surfaces or intracellularly.
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Immunohistochemistry (IHC): Detects vimentin in tissue sections to study its role in pathological conditions such as cancer or fibrosis.
What experimental controls should be included when using Vimentin Antibody, FITC?
To ensure reliable results, researchers should include the following controls:
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Isotype Control: Use an FITC-conjugated antibody of the same isotype but irrelevant specificity to account for non-specific binding.
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Negative Control: Use samples where vimentin expression is absent or knocked down (e.g., vimentin-deficient cells).
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Positive Control: Include samples known to express vimentin robustly, such as fibroblasts or mesenchymal-derived cells.
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Unstained Control: For flow cytometry experiments, include unstained cells to set baseline fluorescence levels.
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Secondary Antibody Control: If a secondary antibody is used, ensure it does not produce non-specific signals.
How can variations in Vimentin expression be quantitatively analyzed?
Quantitative analysis of vimentin expression can be achieved using techniques such as flow cytometry and Western blotting:
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Flow Cytometry: Measures fluorescence intensity corresponding to vimentin-FITC binding on a per-cell basis. This method allows high-throughput analysis of large cell populations .
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Western Blotting: Quantifies vimentin protein levels by densitometric analysis of band intensities normalized to a loading control protein .
In both methods, proper calibration with standards and inclusion of replicates are essential for accurate quantification.
What are the challenges associated with detecting surface-expressed Vimentin?
Surface expression of vimentin is less common than its cytoplasmic localization and may vary depending on cell type and activation state. Challenges include:
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Low Abundance: Surface-expressed vimentin may be present at low levels, requiring sensitive detection methods such as fluorescence-activated cell sorting (FACS) .
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Non-Specific Binding: Surface proteins often interact with multiple ligands; thus, rigorous blocking steps are necessary to minimize background signals.
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Cell Permeabilization: Avoid permeabilization steps when specifically targeting surface-expressed vimentin.
How does Vimentin contribute to epithelial-to-mesenchymal transition (EMT)?
Vimentin serves as a hallmark of EMT, a process where epithelial cells lose their polarity and adhesion properties while gaining migratory and invasive capabilities . During EMT:
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Vimentin expression increases as cells transition from an epithelial to a mesenchymal phenotype.
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It interacts with other cytoskeletal proteins to reorganize cellular architecture.
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This reorganization facilitates enhanced motility and invasiveness, critical for processes like cancer metastasis .
Can Vimentin Antibody, FITC be used to study cancer progression?
Yes, vimentin is extensively studied as a marker for cancer progression due to its role in EMT and metastasis . The antibody can be used to:
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Detect increased vimentin expression in tumor cells undergoing EMT.
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Assess the correlation between vimentin levels and metastatic potential.
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Investigate therapeutic interventions targeting vimentin-mediated pathways.
For instance, flow cytometry can quantify changes in surface-expressed vimentin on cancer cells treated with anti-metastatic agents .
What factors influence the specificity of Vimentin Antibody binding?
The specificity of Vimentin Antibody binding depends on several factors:
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Epitope Recognition: The antibody must recognize a unique epitope on the vimentin protein without cross-reacting with other intermediate filaments.
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Species Reactivity: Verify that the antibody reacts with the species being studied (e.g., human, rat) .
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Experimental Conditions: Optimize antibody concentration, incubation time, and temperature for each application.
How can discrepancies in experimental results involving Vimentin Antibody be resolved?
Discrepancies may arise due to technical or biological factors:
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Technical Factors: Variations in antibody quality, experimental protocols, or detection methods can lead to inconsistent results.
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Biological Factors: Differences in cell type, culture conditions, or disease state may influence vimentin expression.
To resolve these issues:
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Validate results using multiple antibodies targeting different epitopes on vimentin.
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Cross-check findings across complementary techniques like ICC and Western blotting.
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Standardize protocols and include appropriate controls.
Data Table: Comparison of Key Features Across Commonly Used Vimentin Antibodies
| Feature | Clone V9 [FITC] | Clone 7C11-D9 [FITC] | Clone VIM/1937R [FITC] |
|---|---|---|---|
| Species Reactivity | Human, Canine | Human | Human, Rat |
| Applications | ICC, IHC | ICC, Flow Cytometry | Flow Cytometry |
| Epitope Specificity | Cytoplasmic | Peptide-based | Full-length Protein |
| Cross-Reactivity | None Reported | None Reported | None Reported |
| Excitation/Emission Wavelength | 488 nm/520 nm | 495 nm/519 nm | 495 nm/519 nm |
Product Science Overview
Vimentin is a type III intermediate filament protein that is widely expressed in mesenchymal cells. It plays a crucial role in maintaining cell integrity, stabilizing cytoskeletal interactions, and supporting cellular processes such as migration and signaling . Vimentin is encoded by the VIM gene located on chromosome 10 in humans . It is involved in various biological processes, including intermediate filament organization, cellular response to stress, and regulation of gene expression .
During epithelial-mesenchymal transition (EMT), vimentin expression is upregulated, which is essential for processes like wound healing, embryogenesis, and cancer metastasis . Vimentin serves as a marker for mesenchymal cells and is often used in research to study cell differentiation and tumor progression .
Mouse anti-human antibodies are monoclonal antibodies produced by immunizing mice with human antigens. These antibodies are highly specific and are used extensively in research and clinical diagnostics . The development of mouse anti-human antibodies involves the fusion of mouse myeloma cells with spleen cells from immunized mice, creating hybridoma cells that produce the desired antibody .
One of the significant applications of mouse anti-human antibodies is in immunohistochemistry (IHC) and flow cytometry, where they are used to detect specific human proteins in tissue samples . These antibodies can also be conjugated with various labels, such as enzymes or fluorophores, to facilitate detection and quantification .
FITC, or Fluorescein Isothiocyanate, is a fluorescent dye commonly used in biological research for labeling proteins, antibodies, and other biomolecules . FITC has a high absorption rate and excellent fluorescence quantum yield, making it an ideal choice for various fluorescence-based assays . It emits bright yellow-green fluorescence when excited at a wavelength of 494 nm and has an emission peak at 520 nm .
The isothiocyanate group of FITC reacts with primary amines on proteins, forming stable thiourea bonds . This property allows FITC to be used in a wide range of applications, including flow cytometry, fluorescence microscopy, and immunofluorescence .
The combination of vimentin, mouse anti-human antibody, and FITC creates a powerful tool for studying cellular processes and protein localization. The mouse anti-human vimentin antibody specifically binds to human vimentin, allowing researchers to detect and visualize vimentin expression in human cells . When conjugated with FITC, the antibody can be used in fluorescence-based assays to provide detailed insights into vimentin distribution and dynamics within cells .
This conjugate is particularly useful in cancer research, where vimentin expression is often associated with tumor progression and metastasis . By using FITC-labeled mouse anti-human vimentin antibodies, researchers can track changes in vimentin expression during EMT and other critical cellular events .
