Vim Antibody

What is vimentin and why is it significant in cellular research?

Vimentin is a type III intermediate filament protein encoded by the VIM gene. In humans, the canonical protein has 466 amino acid residues and a mass of 53.7 kDa . Vimentin's significance stems from its pivotal role in maintaining cellular architecture and integrity as a major component of the cytoskeleton. It is particularly important in research contexts as a marker for mesenchymal cells and in studying epithelial-mesenchymal transition (EMT), a process critical in development, wound healing, and cancer progression.

Vimentin is localized in the cell membrane, nucleus, and cytoplasm, making it a versatile target for different cellular compartment investigations . Its expression pattern is characteristic, with high expression in fibroblasts, moderate expression in T- and B-lymphocytes, and minimal expression in Burkitt's lymphoma cell lines . This differential expression makes vimentin antibodies particularly valuable for cell type identification and characterization in complex tissue environments.

What are the key applications for vimentin antibodies in laboratory research?

Vimentin antibodies serve multiple research purposes across different experimental platforms. Based on available data, the primary applications include:

The versatility of anti-vimentin antibodies makes them indispensable tools for researchers investigating cellular structure, differentiation processes, and pathological conditions . When selecting an antibody for a specific application, researchers should consider the validation data provided by manufacturers, which typically includes images from multiple applications showing expected staining patterns.

How do I properly select a vimentin antibody for my specific research needs?

The selection of an appropriate vimentin antibody requires consideration of several critical factors:

• Experimental Application: Different applications require antibodies with specific properties. For instance, antibodies for Western blotting may not perform optimally in immunohistochemistry. Review the manufacturer's validation data for your specific application .

• Species Reactivity: Ensure the antibody recognizes vimentin from your experimental species. The search results indicate many antibodies with reactivity to human, mouse, and rat vimentin, while some also cross-react with bovine, chicken, pig, and other species .

• Clonality: Monoclonal antibodies (like V9 and RV202 clones) offer high specificity and reproducibility, while polyclonal antibodies provide broader epitope recognition . For example, the V9 clone shows extensive validation across multiple applications with over 1000 citations .

• Conjugation: Consider whether you need an unconjugated antibody or one conjugated to a fluorophore (FITC, Cy3), enzyme, or biotin based on your detection method .

• Validated Performance: Review user feedback and citation histories. Antibodies with extensive citations and positive user reviews generally indicate reliable performance. For instance, some vimentin antibodies in the search results have hundreds of citations and numerous positive reviews .

When multiple options meet your criteria, selecting antibodies from manufacturers that provide extensive validation data and technical support can significantly reduce troubleshooting time.

How can I optimize vimentin antibody staining in difficult-to-permeabilize tissues?

Optimizing vimentin antibody staining in challenging tissues requires a systematic approach to sample preparation and staining protocols:

Researchers report particular success with the V9 clone in difficult tissue preparations, noting its robust performance even in suboptimally preserved clinical samples .

What strategies can resolve inconsistent vimentin antibody staining patterns in EMT models?

Epithelial-mesenchymal transition (EMT) models often present unique challenges for vimentin detection due to the dynamic nature of protein expression. Researchers have reported several effective strategies to address inconsistent staining:

• Temporal Sampling Design: EMT is a dynamic process, and vimentin expression changes progressively. Implement a comprehensive time-course analysis with sampling at 6-12 hour intervals during the early stages of EMT induction and 24-hour intervals thereafter to capture the full expression profile .

• Dual Marker Approach: Co-stain with both vimentin and epithelial markers (E-cadherin, cytokeratins) to precisely identify cells at various EMT stages. This approach allows for more accurate interpretation of seemingly inconsistent vimentin staining patterns by contextualizing them within the EMT spectrum .

• Clone-Specific Considerations: Different anti-vimentin antibody clones may recognize distinct epitopes that become accessible at different EMT stages. The D21H3 and V9 clones have demonstrated particularly consistent results in EMT models according to user reviews . Validation experiments comparing multiple clones on the same samples can identify the most suitable antibody for your specific EMT model.

• Quantitative Analysis Implementation: Implement digital image analysis with defined intensity thresholds to objectively quantify vimentin expression levels across cell populations. This approach can reveal patterns not immediately apparent through visual inspection alone .

• Fixation Protocol Standardization: EMT studies are particularly sensitive to variations in fixation protocols. Standardize fixation conditions (4% paraformaldehyde for precisely 15 minutes at room temperature) across all experimental timepoints to ensure comparable epitope preservation .

A researcher reported: "We are culturing mesenchymal cells in microfluidic devices. This antibody stains them well" - demonstrating successful application in advanced culture systems .

How can I effectively multiplex vimentin antibodies with other cellular markers?

Successful multiplexing of vimentin with other cellular markers requires careful consideration of antibody compatibility and detection strategies:

• Cross-Reactivity Mitigation: When multiplexing vimentin with other mesenchymal markers (α-SMA, fibronectin), use antibodies raised in different host species to prevent cross-reactivity. For example, pair a mouse monoclonal anti-vimentin with rabbit polyclonal antibodies against other targets .

• Sequential Staining Approach: For challenging combinations, implement sequential staining with complete stripping between rounds using glycine-HCl buffer (pH 2.5) followed by re-blocking. This is particularly effective when antibodies from the same species must be used .

• Spectral Unmixing Techniques: When using fluorescent detection in samples with high autofluorescence, implement spectral unmixing algorithms to accurately separate overlapping signals. This is especially important when studying tissues like lung and liver that naturally emit background fluorescence .

• Clone-Specific Considerations: Certain vimentin antibody clones perform particularly well in multiplexed settings. The RV202 and V9 clones have been extensively validated in multiplex immunofluorescence applications according to citation data .

• Isotype-Matched Controls: Always include appropriate isotype-matched control antibodies for each primary antibody in the multiplex panel to accurately assess background and non-specific binding .

A successful implementation was reported by a researcher who noted: "I used this antibody to check expression of vimentin in A549 lung cancer cells. I did immunofluorescence and the antibody performed very well, with nice and clear signal" .

What are the optimal protocols for detecting post-translationally modified vimentin?

Vimentin undergoes several post-translational modifications (PTMs) that significantly impact its function. Detecting these modified forms requires specialized approaches:

• Phosphorylated Vimentin Detection:

  • Use phospho-specific antibodies targeting known sites (Ser38, Ser55, Ser71, Ser72)

  • Incorporate phosphatase inhibitors (50mM NaF, 5mM sodium pyrophosphate, 1mM Na3VO4) in all buffers during sample preparation

  • Consider lambda phosphatase treatment of control samples to validate phospho-specific staining

• Citrullinated Vimentin Detection:

  • Specialized antibodies against mutated citrullinated vimentin (MCV) are available and have diagnostic value in rheumatoid arthritis research

  • Sample preparation requires calcium-containing buffers to preserve peptidylarginine deiminase (PAD) enzyme activity during fixation

• O-GlcNAcylated Vimentin:

  • Use dual approach combining anti-vimentin immunoprecipitation followed by O-GlcNAc-specific detection

  • Include O-GlcNAcase inhibitors (PUGNAc or Thiamet G) in lysis buffers to preserve this labile modification

• SUMOylated Vimentin:

  • Requires denaturing conditions during lysis (1% SDS with immediate boiling) to preserve SUMO modifications

  • SUMO-specific protease inhibitors (20mM N-ethylmaleimide) are essential in all buffers

These specialized detection methods enable researchers to investigate how PTMs regulate vimentin function in different cellular contexts, particularly during stress responses and cytoskeletal remodeling events.

How can I troubleshoot weak or absent vimentin antibody signals in Western blots?

Weak or absent vimentin signals in Western blots can result from multiple factors. A systematic troubleshooting approach includes:

• Sample Preparation Optimization:

  • Implement a dedicated cytoskeletal extraction buffer (50mM PIPES pH 6.8, 100mM NaCl, 5mM MgCl2, 5mM EGTA, 1% Triton X-100, 0.3M sucrose)

  • Extend sonication time (4-6 pulses of 10 seconds each) to ensure complete solubilization

  • Avoid excessive heat during sample preparation as vimentin can form insoluble aggregates above 65°C

• Transfer Protocol Adjustment:

  • Increase transfer time by 25-50% for high molecular weight proteins like vimentin

  • Reduce methanol concentration in transfer buffer to 10% to enhance elution of large proteins from gels

  • Consider wet transfer systems rather than semi-dry for more complete transfer

• Antibody Selection and Dilution:

  • Test multiple antibody clones if available, as epitope accessibility can vary based on sample preparation

  • Prepare a dilution series (1:500, 1:1000, 1:2000, 1:5000) to identify optimal concentration

  • Extended primary antibody incubation (overnight at 4°C) often improves signal quality

• Detection System Enhancement:

  • Implement signal enhancement systems such as biotin-streptavidin amplification

  • Consider highly sensitive chemiluminescent substrates for weak signals

  • Extended exposure times (up to 30 minutes) may be necessary for low abundance samples

A researcher reported: "I am very satisfied with this antibody. I tested it on endothelial cells (A549) stimulated with TGF-β, which is known to increase vimentin expression. The antibody works well at 1:1000 dilution" . This demonstrates that with proper optimization, reliable results can be achieved even in dynamic expression systems.

What are the best practices for quantifying vimentin expression in heterogeneous tissue samples?

Quantifying vimentin expression in heterogeneous tissues presents unique challenges that require specialized approaches:

• Region-Specific Analysis Strategy:

  • Implement region-of-interest (ROI) analysis with anatomical landmarks as guides

  • Define and apply consistent tissue compartment definitions across all samples

  • Analyze stromal and parenchymal regions separately to account for intrinsic differences in vimentin expression

• Digital Pathology Tools:

  • Utilize color deconvolution algorithms to separate vimentin signal from counterstains

  • Implement machine learning-based segmentation to identify vimentin-positive cells in complex tissues

  • Apply H-score methodology (3× percentage of strongly stained cells + 2× percentage of moderately stained cells + 1× percentage of weakly stained cells) for semi-quantitative assessment

• Normalization Approaches:

  • Include internal reference cell types with known vimentin expression levels

  • Utilize ratiometric analysis comparing vimentin to housekeeping proteins within the same samples

  • Apply tissue microarray (TMA) design principles with methodical sampling of different tissue regions

• Validation Controls:

  • Include both positive control tissues (fibroblast-rich tissues) and negative control tissues (epithelial-rich regions)

  • Implement multiple antibody clones on serial sections to confirm staining patterns

  • Include quantitative Western blot validation for selected samples

A researcher noted: "In the present study, Vimentin expression was detected in venous ulcer. Selection of the right antibody was critical for our differential expression analysis" . This highlights the importance of proper antibody selection for quantitative studies in complex tissue environments.

How do I establish reliable protocols for vimentin antibody-based cell sorting?

Developing robust vimentin antibody-based cell sorting protocols requires careful optimization of several parameters:

• Cell Preparation Protocol:

  • Optimize fixation and permeabilization conditions (0.1% saponin typically preserves scatter properties better than Triton X-100)

  • Implement two-step fixation: brief 2% paraformaldehyde (5 minutes) followed by gentle permeabilization

  • Maintain strict temperature control (4°C) throughout the procedure to preserve cellular integrity

• Antibody Selection Criteria:

  • Choose antibodies specifically validated for flow cytometry applications

  • Select clones with bright signal-to-noise ratios (V9 and RV202 clones are frequently cited for flow applications)

  • Consider directly conjugated antibodies to eliminate secondary antibody variability

• Compensation Strategy:

  • Prepare single-color controls using the same cell type and processing conditions

  • Implement fluorescence-minus-one (FMO) controls to set accurate gates

  • Consider spectral cytometry for complex panels to minimize compensation requirements

• Sorting Parameters:

  • Use low pressure settings (20-30 psi) to maintain cellular integrity

  • Implement doublet discrimination based on FSC-H vs. FSC-A and SSC-H vs. SSC-A

  • Consider index sorting to retain single-cell parameter data for post-sort analysis

• Validation Approach:

  • Analyze sorted fractions using orthogonal methods (qPCR, Western blot)

  • Perform microscopic examination of pre- and post-sorted populations

  • Conduct functional assays to confirm biological relevance of sorted populations

These methodological refinements enable researchers to isolate specific cell populations based on vimentin expression, a valuable approach for studying cellular heterogeneity in complex tissues and cell cultures.

How should vimentin expression be interpreted in cancer research contexts?

Interpreting vimentin expression in cancer contexts requires nuanced analysis that considers multiple factors:

• Cancer Type-Specific Patterns:

  • In carcinomas, vimentin expression often indicates epithelial-mesenchymal transition (EMT) and correlates with increased invasiveness and metastatic potential

  • In sarcomas, vimentin serves as a diagnostic marker but intensity variations may provide prognostic information

  • In melanomas, co-expression with other intermediate filaments helps distinguish melanoma subtypes

• Spatial Distribution Analysis:

  • Focus on invasive fronts where vimentin expression may differ from tumor cores

  • Analyze tumor-stroma boundaries for gradient patterns of expression

  • Evaluate intratumoral heterogeneity by systematic mapping of vimentin-positive regions

• Multi-Marker Interpretation Framework:

  • Integrate vimentin data with epithelial markers (E-cadherin, cytokeratins)

  • Correlate with transcription factors driving EMT (SNAIL, TWIST, ZEB1/2)

  • Analyze in context of stemness markers (CD44, CD133) for cancer stem cell identification

• Quantitative Thresholds:

  • Establish tissue-specific baseline expression from normal controls

  • Implement quartile or decile analysis for patient stratification

  • Consider automated scoring systems to eliminate observer bias

A researcher noted: "I used this antibody to study epithelial mesenchymal transition and fibrosis in A549 and renal cells. It worked great for Western blot" . This demonstrates the utility of vimentin antibodies in studying cancer-related cellular processes.

What are the critical controls needed when studying vimentin in developmental biology research?

Developmental biology research using vimentin antibodies requires rigorous control strategies:

• Temporal Controls:

  • Include systematic developmental stage series to establish normal expression dynamics

  • Implement precise staging controls to account for rapid developmental changes

  • Create developmental expression atlases as reference standards

• Genetic Validation Approach:

  • Utilize vimentin knockout models as negative controls

  • Implement conditional knockout systems for stage-specific validation

  • Consider knockdown approaches with quantitative validation of reduction levels

• Species-Specific Considerations:

  • Verify antibody cross-reactivity with the developmental model species (zebrafish, frog, chicken)

  • Establish species-specific staining patterns from published literature

  • Include cross-species positive controls to confirm antibody functionality

• Tissue Processing Controls:

  • Process all developmental stages identically to eliminate technical variables

  • Include adult tissue positive controls with known vimentin expression

  • Implement batch processing to minimize inter-experimental variation

• Interpretation Framework:

  • Distinguish between normal developmental vimentin expression and pathological patterns

  • Correlate vimentin patterns with known developmental milestones

  • Integrate with lineage tracing data when interpreting dynamic expression changes

These control strategies enable researchers to accurately interpret vimentin expression patterns in developing tissues and distinguish them from experimental artifacts or pathological processes.

How can I distinguish between specific and non-specific vimentin antibody binding in complex tissue samples?

Distinguishing specific from non-specific binding requires implementation of multiple validation strategies:

• Comprehensive Control Panel:

  • Isotype control antibodies matched to primary antibody species, isotype, and concentration

  • Secondary-only controls to assess secondary antibody non-specific binding

  • Absorption controls where primary antibody is pre-incubated with purified antigen

• Validation Across Methodologies:

  • Confirm staining patterns with multiple antibody clones targeting different epitopes

  • Validate IHC/ICC findings with orthogonal methods (Western blot, qPCR)

  • Correlate protein expression with mRNA localization using in situ hybridization

• Pattern Recognition Approach:

  • True vimentin staining follows characteristic filamentous cytoplasmic patterns

  • Non-specific binding often presents as diffuse, non-filamentous staining

  • Cellular compartmentalization should align with known vimentin distribution (primarily cytoplasmic with some nuclear and membrane association)

• Genetic Validation Strategy:

  • Use vimentin-deficient cell lines or tissues as negative controls

  • Implement siRNA knockdown with quantitative validation

  • Consider CRISPR-edited cell lines with complete vimentin knockout

• Signal Intensity Assessment:

  • Compare signal-to-noise ratios across different antibody dilutions

  • Establish extinction curves by serial dilution to identify optimal concentration

  • Implement digital image analysis to quantify background-to-specific signal ratios

A researcher commented: "The vimentin filament network plays a key role in cell architecture and signaling, as well as the regulation of adhesion and migration. This antibody shows the expected filamentous pattern and works well for our studies" . This underscores the importance of pattern recognition in validating specific binding.

How can vimentin antibodies be utilized in biomarker development for fibrotic diseases?

Vimentin antibodies offer significant potential in fibrotic disease biomarker development:

• Quantitative Assessment Protocols:

  • Develop standardized scoring systems specific to each organ system

  • Implement digital pathology algorithms for consistent quantification

  • Correlate vimentin expression with established fibrosis markers (α-SMA, collagen)

• Circulating Biomarker Applications:

  • Detect circulating vimentin fragments using validated antibody pairs in sandwich ELISA

  • Measure citrullinated vimentin as a specific marker for active fibrotic processes

  • Correlate serum vimentin levels with tissue expression patterns

• Multiplex Panel Development:

  • Create disease-specific antibody panels combining vimentin with extracellular matrix proteins

  • Implement spatial profiling to map relationship between vimentin-positive cells and matrix deposition

  • Validate panels across multiple patient cohorts with varying fibrosis severity

• Response Monitoring Applications:

  • Establish baseline-normalized vimentin expression as a treatment response indicator

  • Correlate changes in vimentin-positive cell populations with clinical outcomes

  • Implement vimentin-based assessment in therapeutic clinical trials

• Technical Standardization Approach:

  • Develop reference materials with calibrated vimentin content

  • Establish inter-laboratory validation protocols

  • Create algorithmic approaches for automated quantification

These applications position vimentin antibodies as valuable tools in developing and validating biomarkers for fibrotic diseases across multiple organ systems.

What are the latest methodologies for studying vimentin dynamics in live cell imaging?

Advanced live cell imaging approaches for studying vimentin dynamics combine specialized probes with cutting-edge microscopy techniques:

• Fusion Protein Design Strategies:

  • Implement photoconvertible fluorescent protein tags (mEos, Dendra2) for pulse-chase experiments

  • Utilize split fluorescent protein systems for studying vimentin-vimentin interactions

  • Apply SNAP/CLIP-tag systems for sequential labeling of vimentin subpopulations

• Advanced Microscopy Implementations:

  • Apply structured illumination microscopy (SIM) for enhanced resolution of filament networks

  • Implement lattice light-sheet microscopy for reduced phototoxicity in long-term imaging

  • Utilize fluorescence recovery after photobleaching (FRAP) to measure vimentin turnover rates

• Antibody-Based Live Imaging Approaches:

  • Use cell-permeable nanobodies against vimentin for live-cell applications

  • Apply Fab fragments of anti-vimentin antibodies for reduced interference with function

  • Implement genetically encoded intrabodies for long-term visualization

• Correlative Imaging Strategies:

  • Combine live cell imaging with subsequent immunolabeling of fixed samples

  • Implement live-to-fixed cell correlation using gridded dishes

  • Apply correlated light and electron microscopy (CLEM) for ultrastructural context

• Computational Analysis Frameworks:

  • Develop filament tracking algorithms for automated dynamics analysis

  • Implement tensor-based analysis for filament orientation quantification

  • Apply optical flow analysis for measuring global network reorganization

These methodological advancements enable researchers to study the dynamic behavior of vimentin in living cells with unprecedented spatial and temporal resolution, providing new insights into its functions in cellular processes.