ITGAV Antibody, FITC conjugated

What is ITGAV and what role does it play in cellular biology?

ITGAV (Integrin Alpha V) is a transmembrane glycoprotein that forms heterodimers with various beta integrin subunits to create functional integrin complexes. These complexes are critical for cell-cell and cell-matrix adhesion, playing essential roles in cellular migration, invasion, proliferation, and survival. ITGAV expression has been associated with tumor progression in several cancer types, including breast cancer, where overexpression correlates with poor relapse-free survival . The protein is present in various cellular structures, including as light and heavy chains, and functions through interaction with the extracellular matrix to regulate critical cellular processes. In breast cancer specifically, ITGAV contributes to tumorigenesis and metastasis through upregulation of PXN (paxillin) .

How do FITC-conjugated antibodies function in flow cytometry?

FITC (Fluorescein Isothiocyanate) conjugated antibodies allow for direct visualization of target antigens through fluorescence detection. In flow cytometry, these antibodies bind specifically to their target proteins (such as ITGAV) on the cell surface, enabling quantitative assessment of protein expression. When excited by the blue laser (488 nm), FITC emits green fluorescence at approximately 520 nm .

For optimal flow cytometry results, researchers should:

• Use pre-titrated antibody concentrations (typically 5 μL or 0.25 μg per test for 10^5-10^8 cells)

• Include appropriate negative controls and compensation controls

• Ensure sample preparation includes proper blocking to reduce non-specific binding

• Analyze data using appropriate gating strategies based on control samples

When specifically working with integrin antibodies, it's important to recognize that some antibody clones may possess activating activity for their target integrins, as noted with certain CD29 antibodies , which should be considered when interpreting results.

What are the recommended dilutions and applications for ITGAV antibodies?

While specific dilutions for FITC-conjugated ITGAV antibodies aren't directly addressed in the search results, we can reference standard applications and dilutions for ITGAV antibodies in general:

For flow cytometry applications with fluorochrome-conjugated antibodies (such as FITC conjugates), researchers should typically follow manufacturer recommendations for the specific clone, but a starting point of 5 μL (0.25 μg) per test in a final volume of 100 μL is often appropriate . Cell numbers can range from 10^5 to 10^8 cells per test, with specific optimization required for different applications and cell types.

How should researchers validate ITGAV antibody specificity?

Validating antibody specificity is critical for reliable research outcomes. For ITGAV antibodies, consider these methodological approaches:

• Positive and negative tissue/cell controls: Verify expression in known ITGAV-positive samples such as human placenta tissue, HUVEC cells, A549 cells, and MCF-7 cells .

• Cross-reactivity testing: Check specificity across species. Most ITGAV antibodies show reactivity with human samples, while cross-reactivity with other species varies by clone .

• Knockdown/knockout validation: Use siRNA or CRISPR to reduce ITGAV expression and confirm antibody signal reduction. This has been demonstrated in studies where ITGAV silencing significantly reduced detection in immunofluorescence staining of MB231 cells .

• Multiple detection methods: Confirm expression using complementary techniques (e.g., if using IHC, validate with Western blot).

• Immunofluorescence microscopy: Confirm appropriate cellular localization patterns consistent with ITGAV's known distribution in cellular membranes.

How does ITGAV expression correlate with cancer progression and patient outcomes?

ITGAV expression has significant prognostic implications in cancer research. Studies indicate:

• Prognostic value: Overexpression of ITGAV in primary tumors strongly correlates with poor relapse-free survival (RFS) in luminal B, HER2, and triple-negative breast cancer patients .

• Tumor vs. normal tissue: Immunohistochemistry analysis demonstrates significantly higher ITGAV expression in breast tumor tissue compared to adjacent normal tissue .

• Functional significance: ITGAV silencing inhibits cell proliferation, invasion, migration, and colony formation in breast cancer cell lines (MB231, T47D), demonstrating its mechanistic role in tumor progression .

• Therapeutic targeting: The ITGAV antagonist cilengitide significantly reduces lung metastasis in metastatic breast cancer animal models and inhibits cell proliferation in vitro, suggesting ITGAV as a promising therapeutic target .

• Molecular mechanism: ITGAV promotes breast cancer progression through upregulation of PXN, contributing to enhanced cell survival, proliferation, and invasiveness .

These findings suggest ITGAV expression levels could serve as useful predictors of patient treatment responses and outcomes, particularly in specific breast cancer subtypes.

What methodological approaches can researchers use to measure ITGAV-mediated functional effects?

To investigate ITGAV's role in cellular functions, researchers can employ several methodological approaches:

• Knockdown studies: siRNA-mediated ITGAV silencing can be confirmed through qPCR, western blot, and immunofluorescence staining. This approach has revealed ITGAV's role in cell proliferation across multiple breast cancer cell lines (MB231, T47D, MCF7, MB468, SKBR3) .

• Proliferation assays: MTT or similar assays can quantify how ITGAV silencing or antagonism (e.g., with cilengitide) affects cell proliferation rates .

• Colony formation assays: Soft agar, anchorage-free cultures can assess how ITGAV affects clonogenic potential and cellular independence .

• Invasion/migration assays: Transwell or scratch assays can evaluate ITGAV's impact on cellular invasiveness .

• Stem-like phenotype characterization: Flow cytometry can analyze markers like CD44high/CD24low and ALDH activity following ITGAV manipulation to assess effects on cancer stemness .

• In vivo metastasis models: Animal studies using cilengitide or genetic ITGAV manipulation can evaluate effects on tumor growth and metastatic spread .

• Molecular pathway analysis: Investigate downstream targets affected by ITGAV, such as BCL2 and PXN, using western blot or qPCR to understand mechanistic details .

These complementary approaches provide comprehensive insights into ITGAV's functional roles in cancer biology.

What are the technical considerations for optimizing FITC-conjugated antibody performance?

When working with FITC-conjugated antibodies, including those targeting ITGAV, researchers should consider these technical factors:

• Spectral characteristics: FITC excites at 488 nm and emits at approximately 520 nm, requiring appropriate laser configuration and filter sets on flow cytometers .

• Photobleaching: FITC is relatively susceptible to photobleaching. Minimize light exposure during sample preparation and storage.

• pH sensitivity: FITC fluorescence is pH-dependent, with optimal signal at slightly alkaline pH. Buffer selection affects signal intensity.

• Purification quality: High-quality FITC-conjugated antibodies undergo size-exclusion chromatography to remove unconjugated antibody and free fluorochrome, ensuring specific signal .

• Conjugation ratio: The fluorophore-to-protein ratio affects brightness and potential antibody binding interference.

• Autofluorescence management: FITC emission overlaps with cellular autofluorescence, particularly in fixed cells. Include appropriate negative controls.

• Multicolor panel design: When designing multicolor panels, account for FITC spectral overlap with other fluorochromes (particularly PE) and implement proper compensation controls.

• Storage conditions: FITC conjugates typically require storage at 4°C protected from light. Avoid repeated freeze-thaw cycles.

• Signal amplification: For low-abundance targets, consider sequential staining strategies or higher-brightness alternatives to FITC.

How can researchers troubleshoot non-specific binding when using ITGAV antibodies?

When encountering non-specific binding with ITGAV antibodies, researchers should implement these troubleshooting strategies:

• Titration optimization: Test a range of antibody dilutions (1:200-1:10000 depending on application) to identify the optimal signal-to-noise ratio .

• Blocking optimization: Use species-appropriate serum or protein blockers (BSA, casein) at sufficient concentrations and incubation times.

• Wash protocol revision: Increase wash duration or number of washes with appropriate detergent concentration to reduce non-specific binding.

• Sample preparation refinement: Ensure proper fixation and permeabilization protocols that preserve epitope accessibility while maintaining cellular integrity.

• Cross-reactivity assessment: Verify known cross-reactivity patterns. For example, some ITGAV antibodies react with human, porcine, and canine samples but not with mouse samples .

• Epitope considerations: Select antibodies recognizing appropriate epitopes based on your application. Some antibodies target specific amino acid regions (e.g., AA 297-380 for certain ITGAV antibodies) .

• Isotype controls: Include appropriate isotype controls (such as IgG1 for many monoclonal antibodies) to distinguish specific from non-specific binding .

• Absorption controls: Pre-absorb antibodies with the immunizing peptide when available to confirm specificity.

• Alternative detection methods: If persistent issues occur with one detection method, validate findings using complementary techniques.

What emerging applications exist for ITGAV targeting in cancer research?

Recent research reveals several promising applications for ITGAV targeting in cancer research:

• Therapeutic targeting: The ITGAV antagonist cilengitide has shown significant efficacy in reducing lung metastasis in breast cancer models, highlighting its potential as a therapeutic approach for metastatic disease .

• Biomarker development: ITGAV expression levels demonstrate strong potential as prognostic biomarkers for predicting relapse-free survival, particularly in luminal B, HER2, and triple-negative breast cancer patients .

• Combinatorial therapy approaches: ITGAV targeting combined with conventional chemotherapies may enhance treatment efficacy by inhibiting both primary tumor growth and metastatic spread.

• Personalized medicine applications: Patients with tumors showing high ITGAV expression may benefit from targeted anti-ITGAV therapies, allowing for more personalized treatment approaches .

• Molecular pathway targeting: Research has identified that ITGAV promotes cancer progression through PXN upregulation, suggesting potential for dual-targeting approaches focusing on both ITGAV and its downstream effectors .

• Cell-based immunotherapies: Manipulating ITGAV expression or function may enhance immune cell trafficking and tumor infiltration in immunotherapy approaches.

• Circulating tumor cell detection: ITGAV antibodies could potentially improve detection and characterization of circulating tumor cells in liquid biopsies.

These emerging applications demonstrate the expanding importance of ITGAV in cancer research beyond basic expression studies, highlighting its potential in translational oncology applications.

What is the recommended protocol for using FITC-conjugated antibodies in flow cytometry?

For optimal results with FITC-conjugated antibodies in flow cytometry applications, researchers should follow this methodological approach:

• Sample preparation:

  • Harvest cells (10^5-10^8 cells per test) and wash twice in flow cytometry buffer (PBS with 1-2% FBS or BSA)

  • Resuspend cells in 100 μL buffer per test

  • For intracellular targets, use appropriate fixation and permeabilization reagents

• Antibody staining:

  • Add 5 μL (0.25 μg) of pre-titrated FITC-conjugated antibody per test

  • Include appropriate controls (unstained, isotype, and single-color compensation controls)

  • Incubate for 15-30 minutes at appropriate temperature (typically 4°C or room temperature) protected from light

  • Wash twice with buffer to remove unbound antibody

• Instrument setup:

  • Configure flow cytometer with 488 nm laser excitation

  • Use appropriate bandpass filters for FITC detection (~525/40 nm)

  • Set PMT voltages using unstained controls

  • If using multiple fluorochromes, perform compensation using single-color controls

• Data acquisition and analysis:

  • Collect sufficient events (typically 10,000-50,000 per sample)

  • Apply appropriate gating strategies based on forward/side scatter and viability markers

  • Analyze data using appropriate statistical methods, comparing to controls

• Special considerations for ITGAV analysis:

  • For cell surface markers, maintain cells in non-permeabilized state

  • Consider the activation state of integrins, as some antibody clones can induce integrin activation

  • For dual staining with apoptosis markers, follow specific protocols for annexin V-FITC and PI staining

How do different fixation methods affect ITGAV antibody staining in immunohistochemistry?

Different fixation methods can significantly impact ITGAV antibody staining in immunohistochemistry applications:

• Formalin fixation (FFPE tissues):

  • Most common method for clinical samples

  • Requires antigen retrieval for optimal ITGAV detection

  • For ITGAV antibodies, heat-induced epitope retrieval using TE buffer (pH 9.0) is recommended, with citrate buffer (pH 6.0) as an alternative

  • Typical antibody dilutions range from 1:200-1:800 for IHC applications

  • Has been successfully used to detect differential ITGAV expression between tumor and adjacent normal tissue in breast cancer studies

• Frozen tissue sections:

  • Preserves native epitopes better than FFPE

  • Typically requires acetone or methanol fixation

  • May provide better staining with some ITGAV antibody clones

  • Often used for double-immunofluorescence studies

  • Some ITGAV antibodies are specifically validated for IHC on frozen sections

• Cell preparations:

  • For cultured cells, paraformaldehyde (2-4%) fixation is common

  • Methanol fixation may improve nuclear antigen accessibility but can affect membrane protein detection

  • Immunofluorescence staining of cultured cells has been successfully used to confirm ITGAV knockdown

• Tissue preparation considerations:

  • Section thickness (typically 5-6 μm) affects staining quality

  • Dewaxing and rehydration protocols influence antigen accessibility

  • Blocking protocols (typically 1 hour at room temperature) are crucial for reducing background

• Detection systems:

  • DAB detection kits provide good signal-to-noise ratios for ITGAV

  • For fluorescence detection, appropriate secondary antibodies must be selected based on the primary antibody host species

These methodological considerations are essential for obtaining specific and sensitive ITGAV detection in different sample types.

How can ITGAV antibodies be used to investigate cancer stem cell properties?

ITGAV antibodies provide valuable tools for investigating cancer stem cell (CSC) properties through several methodological approaches:

• Flow cytometric analysis of stem-like phenotypes:

  • Cancer stem cells can be characterized using surface markers like CD44high/CD24low and ALDH enzymatic activity

  • ITGAV antibodies can be combined with these markers in multicolor flow cytometry to evaluate correlation between ITGAV expression and stemness

  • For such analyses, cells are typically stained with fluorescently-conjugated anti-CD24 and anti-CD44 for 20 minutes at room temperature

  • ALDH activity is assessed by incubating cells with ALDEFLUOR assay buffer containing ALDH substrate for 40 minutes at 37°C, with DEAB-stained aliquots as negative controls

• Self-renewal assessment:

  • ITGAV silencing has been shown to inhibit self-renewal of breast cancer cell lines

  • Sphere formation assays in low-attachment conditions can evaluate how ITGAV affects CSC self-renewal capacity

  • Colony formation in soft agar following ITGAV manipulation provides insights into anchorage-independent growth, a CSC characteristic

• Therapeutic resistance studies:

  • CSCs often display enhanced therapeutic resistance

  • ITGAV expression analysis before and after treatment can reveal selection for ITGAV-expressing CSC populations

  • Decreased expression of ITGAV correlates with acquiring multidrug resistance in some tumor cells

• In vivo studies:

  • Limited dilution assays following ITGAV manipulation can assess tumor-initiating capacity in vivo

  • Immunohistochemical analysis of xenograft tumors using anti-ITGAV and anti-Ki67 antibodies can evaluate stemness and proliferation relationships

These approaches provide comprehensive insights into how ITGAV influences cancer stem cell properties, with important implications for understanding therapeutic resistance and tumor recurrence.

What methods are recommended for quantifying ITGAV expression in tissue microarrays?

For accurate quantification of ITGAV expression in tissue microarrays (TMAs), researchers should implement the following methodological approaches:

• Optimal staining protocol:

  • Use formalin-fixed, paraffin-embedded tissue sections (typically 6 μm)

  • Perform dewaxing and rehydration with ethanol

  • Conduct antigen retrieval using citrate buffer solution (10 mM) or TE buffer (pH 9.0)

  • Block with hydrogen peroxide (3%) followed by blocking solution for 1 hour at room temperature

  • Incubate with primary anti-ITGAV antibody overnight at 4°C (typically 1:200-1:800 dilution)

  • Apply appropriate secondary antibodies for 30 minutes

  • Visualize using DAB detection kit

• Scoring systems:

  • Implement standardized scoring based on staining intensity (0-3+)

  • Consider percentage of positive cells (0-100%)

  • Calculate H-score (intensity × percentage) for semi-quantitative analysis

  • Use digital image analysis software for more objective quantification

• Controls and validation:

  • Include positive controls (human placenta tissue, breast cancer tissue, liver cancer tissue, stomach cancer tissue)

  • Include negative controls (antibody diluent without primary antibody)

  • Use normal adjacent tissue as internal control when available

  • Validate findings with alternative detection methods like Western blot

• Data analysis considerations:

  • Associate ITGAV expression with clinical parameters and outcomes

  • Studies have shown ITGAV overexpression correlates with poor relapse-free survival in specific breast cancer subtypes

  • Compare expression between tumor and adjacent normal tissue to establish differential expression

  • Consider subgroup analyses based on tumor type, grade, and molecular subtype

• Digital pathology approaches:

  • Use whole slide imaging and automated analysis algorithms

  • Standardize image acquisition parameters

  • Apply machine learning algorithms for pattern recognition and quantification

  • Validate computer-assisted scoring against manual pathologist assessment

These methodological approaches ensure reliable quantification of ITGAV expression in TMAs for biomarker development and prognostic studies.

How might FITC-conjugated ITGAV antibodies be used in multiplex imaging systems?

FITC-conjugated ITGAV antibodies offer significant potential in multiplex imaging systems through these methodological approaches:

• Spectral unmixing platforms:

  • FITC's emission spectrum (peak ~520 nm) can be effectively separated from other fluorophores

  • Advanced spectral imaging systems can distinguish FITC from spectrally similar fluorophores

  • This allows simultaneous detection of ITGAV alongside other markers in the same tissue section

• Sequential multiplexing approaches:

  • FITC signal can be captured and then quenched or stripped

  • Additional rounds of staining with different antibodies can follow

  • This cyclic immunofluorescence approach allows visualization of numerous markers on a single sample

• Antibody conjugation strategies:

  • FITC conjugation typically involves reaction with lysine residues under optimum conditions

  • Unconjugated antibody and free fluorochrome are removed by size-exclusion chromatography

  • Custom conjugation kits allow researchers to create FITC-labeled ITGAV antibodies from unconjugated stocks

• Complementary marker combinations:

  • FITC-conjugated ITGAV antibodies can be paired with markers for:

    • Extracellular matrix components (to study integrin-matrix interactions)

    • Cell proliferation markers (Ki67) to correlate ITGAV with proliferative capacity

    • Cancer stem cell markers (CD44/CD24) to investigate relationships with stemness

    • Invasion markers to explore metastatic potential

• Spatial analysis applications:

  • Multiplex imaging allows visualization of ITGAV distribution relative to tumor architecture

  • Analysis of tumor-stroma interface can reveal invasion-related ITGAV expression patterns

  • Single-cell spatial analysis can identify cellular neighborhoods with distinct ITGAV expression

These applications demonstrate the versatility of FITC-conjugated ITGAV antibodies in advancing our understanding of spatial relationships between ITGAV expression and tumor biology.

What are the methodological considerations for developing therapeutic strategies targeting ITGAV?

Developing effective therapeutic strategies targeting ITGAV requires careful methodological considerations:

• Target validation approaches:

  • Genetic silencing using siRNA has demonstrated ITGAV as a viable therapeutic target in breast cancer

  • ITGAV knockdown inhibits cell proliferation, invasion, and self-renewal of breast cancer cell lines

  • Treatment with the ITGAV antagonist cilengitide significantly reduces lung metastasis in animal models

• Patient stratification strategies:

  • ITGAV overexpression correlates with poor relapse-free survival in luminal B, HER2, and triple-negative breast cancer patients

  • ITGAV expression levels may serve as useful predictors of patient treatment responses

  • IHC detection of ITGAV in tumor versus normal tissue can identify patients likely to benefit from anti-ITGAV therapies

• Therapeutic approaches:

  • Small molecule antagonists (like cilengitide) have shown efficacy in preclinical models

  • Blocking antibodies targeting ITGAV-containing integrin complexes

  • Peptide-based inhibitors mimicking integrin binding regions

  • RNA interference approaches for ITGAV silencing

• Mechanism of action studies:

  • ITGAV promotes breast cancer progression through upregulation of PXN (paxillin)

  • Silencing of ITGAV alters expression of BCL2, affecting apoptosis pathways

  • Understanding these mechanisms is crucial for developing combination therapies

• Combination therapy design:

  • ITGAV targeting combined with conventional chemotherapies

  • Dual targeting of ITGAV and its downstream effectors like PXN

  • Integration with immunotherapeutic approaches

• Safety considerations:

  • ITGAV is broadly expressed on various normal cell types

  • Therapeutic window assessment is critical

  • Targeted delivery systems may improve specificity

• Clinical trial design:

  • Biomarker-driven patient selection based on ITGAV expression

  • Appropriate endpoints (progression-free survival, metastasis-free survival)

  • Pharmacodynamic markers to confirm target engagement

These methodological considerations provide a framework for developing and evaluating ITGAV-targeted therapeutic strategies with potential to address both primary tumors and metastatic disease.