ITGB3 Antibody

What is ITGB3 and why is it important in research?

Integrin beta-3 (ITGB3) is a transmembrane protein that facilitates cell-extracellular matrix interactions or focal adhesions. ITGB3 forms heterodimeric complexes with different alpha integrin subunits, most notably with integrin alpha-IIb (CD41) forming the GpIIb/GpIIIa complex (CD41/CD61) and with integrin alpha-V forming the CD51/CD61 complex. The CD41/CD61 complex appears early in megakaryocyte maturation and serves as a receptor for von Willebrand factor, fibrinogen, fibronectin, vitronectin, and thrombospondin, playing a central role in platelet activation and aggregation. The CD51/CD61 complex is implicated in tumor metastasis and adenoviral infection. ITGB3's involvement in these critical physiological and pathological processes makes it an important research target in hematology, oncology, and cell biology .

What are the different types of ITGB3 antibodies available for research?

Researchers can utilize several types of ITGB3 antibodies, each with specific advantages:

These antibodies recognize different epitopes within ITGB3, with some targeting the individual subunit and others (such as IPI-ITGAV/ITGB3.7) specifically recognizing the heterodimeric complex. Selection should be based on specific experimental requirements and the biological question being addressed .

What applications are ITGB3 antibodies validated for?

ITGB3 antibodies have been validated for multiple research applications:

• Immunohistochemistry-Paraffin (IHC-P): Most commercial antibodies are validated for IHC-P at dilutions of 1-2 μg/ml, enabling visualization of ITGB3 expression in tissue sections .

• Western Blot (WB): Detects ITGB3 protein (typically at 105 kDa / 90 kDa) in cell or tissue lysates .

• Immunoprecipitation (IP): For isolation of ITGB3 protein complexes from cellular extracts .

• Flow Cytometry: Enables detection of ITGB3 on cell surfaces, particularly useful for studying platelets and megakaryocytes .

• Immunofluorescence: Recommended for visualization of ITGB3 in rodent tissues .

When selecting an antibody, researchers should verify that it has been specifically validated for their application of interest and target tissue or cell type .

How should I design experiments to investigate ITGB3-mediated cell adhesion?

When investigating ITGB3-mediated cell adhesion, consider a multi-faceted experimental approach:

• Adhesion assays: Coat plates with ITGB3 ligands (fibronectin, vitronectin, fibrinogen) and assess adhesion of cells expressing ITGB3. Compare with function-blocking anti-ITGB3 antibodies like IPI-ITGAV/ITGB3.7 that specifically block ligand binding .

• Specificity controls: Include control substrates that engage other integrins (collagen for α1β1/α2β1) to confirm specificity of ITGB3-mediated adhesion.

• Activation studies: Compare adhesion under different activation conditions, as integrins exist in active and inactive conformations. Include Mn²⁺ treatment which induces integrin activation.

• Antibody validation: Use multiple anti-ITGB3 antibodies targeting different epitopes to confirm findings. Consider using both antibodies against ITGB3 alone and antibodies against specific heterodimeric complexes (αVβ3) .

• Functional readouts: Beyond simple adhesion, measure downstream signaling events (FAK phosphorylation, cytoskeletal reorganization) to fully characterize the functional consequences of ITGB3-mediated adhesion.

This comprehensive approach will provide robust data on ITGB3's role in cell adhesion while controlling for technical and biological variables.

What are best practices for immunohistochemical detection of ITGB3 in tissue samples?

For optimal immunohistochemical detection of ITGB3 in tissue samples:

• Fixation and processing: Use 10% neutral buffered formalin fixation for 24-48 hours followed by paraffin embedding. Over-fixation can mask epitopes.

• Antigen retrieval: Heat-induced epitope retrieval (HIER) in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is generally effective for ITGB3 detection.

• Antibody selection and dilution: For paraffin sections, both mouse monoclonal (ITGB3/2145) and rabbit monoclonal (ITGB3/2166R) antibodies perform well at dilutions of 1-2 μg/ml. Optimize dilution for each tissue type .

• Positive controls: Include platelet-rich tissues or megakaryocytes, which strongly express ITGB3. The antibody detects platelets in blood and bone marrow smears and megakaryocytes in frozen sections .

• Negative controls: Include antibody isotype controls (Mouse IgG2b or Rabbit IgG) to account for non-specific binding .

• Detection systems: For mouse primary antibodies, use anti-mouse secondary antibodies; for rabbit primaries, use anti-rabbit secondaries. For the chimeric antibody with rabbit constant domains, an anti-rabbit IgG secondary is recommended .

• Counterstaining: Hematoxylin provides good nuclear contrast without obscuring membrane/cytoplasmic ITGB3 staining.

Following these guidelines will maximize specificity and sensitivity while minimizing background in ITGB3 immunohistochemistry.

How can I distinguish between different integrin heterodimers containing ITGB3?

Distinguishing between different ITGB3-containing heterodimers (primarily αIIbβ3 and αVβ3) requires strategic experimental design:

• Heterodimer-specific antibodies: Use antibodies that specifically recognize the heterodimeric complexes rather than individual subunits. For example, the chimeric recombinant antibody IPI-ITGAV/ITGB3.7 specifically targets the αVβ3 heterodimer .

• Co-immunoprecipitation: Immunoprecipitate with anti-ITGB3 antibody followed by Western blotting for αIIb or αV to determine which heterodimers are present.

• Cell-type context: Leverage biological context - αIIbβ3 is predominantly expressed on platelets and megakaryocytes, while αVβ3 has broader expression including endothelial cells and certain tumor cells .

• Functional assays: Design assays based on heterodimer-specific functions:

  • αIIbβ3 specifically binds fibrinogen during platelet aggregation

  • αVβ3 preferentially binds vitronectin in many cell types

• Ligand binding specificity: Use RGD-containing ligands with different affinities for each heterodimer complex, combined with blocking antibodies.

• Co-localization studies: Perform dual immunofluorescence with antibodies against ITGB3 and either αIIb or αV to visualize heterodimer distribution.

This multi-modal approach enables reliable discrimination between different ITGB3-containing heterodimers in experimental systems .

What are common sources of false positives/negatives when using ITGB3 antibodies?

When working with ITGB3 antibodies, researchers should be aware of several potential sources of false results:

False Positives:

• Cross-reactivity with other beta integrins: Some antibodies may cross-react with other beta integrins due to structural similarities. Always verify specificity through appropriate controls .

• Non-specific binding: Particularly in IHC applications, endogenous peroxidases, biotin, or Fc receptor-mediated binding can cause background. Use appropriate blocking steps and isotype controls (Mouse IgG2b [PLRV219/MPC-11] for mouse antibodies or Rabbit IgG for rabbit antibodies) .

• Platelets in tissue samples: As platelets strongly express ITGB3, contaminating platelets in tissue samples may be misinterpreted as tissue expression. Examine morphology carefully .

False Negatives:

• Epitope masking: Fixation can mask epitopes, particularly in formalin-fixed tissues. Ensure proper antigen retrieval - both mouse monoclonal (ITGB3/2145) and rabbit monoclonal (ITGB3/2166R) antibodies require optimized retrieval methods .

• Inactive conformation: ITGB3 exists in active and inactive conformations with different epitope accessibility. Some antibodies may preferentially recognize one conformation .

• Storage degradation: Antibody activity can decrease with repeated freeze/thaw cycles. For long-term storage, aliquot and store at -20°C, avoiding repeated freezing and thawing .

• Suboptimal dilution: At too high concentrations, prozone effects may occur; at too low concentrations, sensitivity is compromised. Titrate antibodies for each application (recommended starting range: 1-2 μg/ml for IHC-P) .

To mitigate these issues, always include positive and negative controls, and consider using multiple antibodies targeting different epitopes to confirm findings.

How do I interpret contradictory ITGB3 expression data between different detection methods?

When faced with contradictory ITGB3 expression data across different detection methods, apply this systematic troubleshooting approach:

• Method-specific considerations:

  • IHC vs. Flow cytometry: IHC detects both surface and intracellular proteins, while flow primarily detects surface expression. Discrepancies may reflect differences in protein localization rather than total expression .

  • Western blot vs. IHC/Flow: WB detects denatured protein and reflects total protein content, whereas IHC/Flow detect native conformations and may be influenced by epitope accessibility .

• Antibody properties:

  • Different epitopes: Antibodies recognizing different epitopes may yield different results if epitopes are differentially accessible in various contexts. Compare the epitope regions (e.g., some antibodies target aa 385-490 of human ITGB3) .

  • Conformational sensitivity: Some antibodies preferentially recognize activated integrins. The chimeric antibody IPI-ITGAV/ITGB3.7 specifically recognizes the αVβ3 heterodimer conformation .

• Biological variables:

  • Heterodimer composition: ITGB3 forms different heterodimers (αIIbβ3, αVβ3) with distinct expression patterns. Some antibodies might preferentially detect specific heterodimers .

  • Activation state: ITGB3 undergoes conformational changes upon activation, potentially affecting epitope accessibility .

• Technical validation steps:

  • Perform titration experiments for each method

  • Use multiple antibodies targeting different epitopes

  • Include positive controls (platelets or megakaryocytes)

  • Verify specificity with genetic approaches (knockdown/knockout)

When properly contextualized, seemingly contradictory results often reveal important biological insights about protein conformation, localization, or complex formation rather than technical errors.

What approaches can resolve weak or inconsistent ITGB3 staining in immunohistochemistry?

When encountering weak or inconsistent ITGB3 staining in immunohistochemistry, systematically address these potential factors:

• Fixation optimization:

  • Overfixation can mask epitopes; optimize fixation time (typically 24-48 hours in 10% neutral buffered formalin)

  • Consider testing different fixatives if working with difficult samples

• Antigen retrieval enhancement:

  • Test multiple retrieval methods (heat-induced vs. enzymatic)

  • Optimize buffer conditions (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • Extend retrieval times for challenging samples

• Signal amplification strategies:

  • Implement polymeric detection systems rather than standard avidin-biotin complexes

  • Consider tyramide signal amplification for very low abundance targets

  • For fluorescent detection, use brighter fluorophores or amplification systems

• Antibody optimization:

  • Test different antibody clones (ITGB3/2145 vs. ITGB3/2166R)

  • Optimize antibody concentration (recommended range: 1-2 μg/ml for IHC-P)

  • Extend primary antibody incubation time (overnight at 4°C)

  • Consider carrier-free antibody formulations to eliminate potential interference from BSA or azide

• Technical considerations:

  • Ensure sections are not too thick (optimal: 4-5 μm)

  • Minimize time between sectioning and staining

  • Use freshly prepared reagents

• Controls and validation:

  • Always include positive control tissue known to express ITGB3 (platelets, megakaryocytes)

  • Use multiple antibodies targeting different epitopes

  • Consider phosphatase-labeled secondary antibodies if endogenous peroxidase activity is problematic

By systematically addressing these factors, most weak or inconsistent ITGB3 staining issues can be resolved to produce reliable, reproducible results.

How can ITGB3 antibodies be utilized in studies of platelet function and megakaryoblastic leukemia?

ITGB3 antibodies offer powerful tools for investigating platelet function and megakaryoblastic leukemia through multiple sophisticated approaches:

• Diagnostic applications in leukemia:

  • Both mouse monoclonal (ITGB3/2145) and rabbit monoclonal (ITGB3/2166R) antibodies are specifically noted to be useful for classification of megakaryoblastic leukemia .

  • Combine with other megakaryocytic markers (CD41, CD42b) for precise lineage classification in acute leukemias.

  • ITGB3/CD61 detection in bone marrow biopsies helps distinguish megakaryoblastic from other leukemia subtypes.

• Functional studies of platelet activation:

  • Use flow cytometry with anti-ITGB3 antibodies to quantify activation-dependent conformational changes in αIIbβ3.

  • Combine with activation markers (P-selectin, phosphatidylserine exposure) to correlate ITGB3 conformational changes with platelet activation states.

  • Employ function-blocking antibodies (like IPI-ITGAV/ITGB3.7 for αVβ3) to assess integrin-specific contributions to platelet aggregation and adhesion .

• Mechanistic studies of integrin signaling:

  • Use phospho-specific antibodies against downstream signaling molecules alongside ITGB3 antibodies to correlate integrin engagement with signaling cascades.

  • Apply conformation-specific ITGB3 antibodies to distinguish active vs. inactive integrin pools during platelet activation.

• Therapeutic development:

  • Screen potential anti-thrombotic compounds using ITGB3 antibodies to assess effects on integrin activation.

  • Evaluate the effects of existing anti-platelet therapies on ITGB3 activation and clustering.

This multifaceted approach leverages the specificity of ITGB3 antibodies to advance both basic research and clinical applications in platelet biology and megakaryoblastic malignancies .

What methodological approaches enable studying ITGB3's role in tumor metastasis?

Investigating ITGB3's role in tumor metastasis requires sophisticated methodological approaches combining molecular, cellular, and in vivo techniques:

• Expression profiling in metastatic progression:

  • Perform systematic IHC analysis of ITGB3 expression across primary tumors and matched metastatic lesions using antibodies like ITGB3/2145 or ITGB3/2166R .

  • Quantify heterodimer-specific expression (αVβ3 vs. αIIbβ3) using specialized antibodies like IPI-ITGAV/ITGB3.7 to determine which complex predominates in different stages .

  • Correlate expression patterns with clinical outcomes to establish prognostic significance.

• Functional interrogation in cellular models:

  • Employ function-blocking antibodies (such as the RGD-mimetic chimeric antibody IPI-ITGAV/ITGB3.7) to disrupt ITGB3-mediated adhesion and signaling .

  • Combine with 3D invasion assays, transendothelial migration models, and matrix degradation assays to assess specific contributions to metastatic processes.

  • Use live-cell imaging with fluorescently-labeled antibodies to track ITGB3 dynamics during invasion and migration.

• Molecular mechanism investigation:

  • Use co-immunoprecipitation with anti-ITGB3 antibodies to identify interaction partners specific to metastatic cells.

  • Combine with phospho-proteomics to map ITGB3-dependent signaling networks activated during metastasis.

• In vivo metastasis models:

  • Utilize antibodies validated for mouse reactivity to study ITGB3 in murine metastasis models .

  • Implement intravital imaging with fluorescently-labeled antibody fragments to visualize ITGB3 dynamics during metastatic colonization.

  • Test therapeutic potential of function-blocking anti-ITGB3 antibodies in preventing metastatic spread.

• Clinical correlation studies:

  • Develop tissue microarrays from patient samples spanning different metastatic stages for high-throughput ITGB3 expression analysis.

  • Correlate ITGB3 expression/activation with response to specific treatments to identify predictive biomarkers.

These methodological approaches collectively provide a comprehensive framework for dissecting ITGB3's multifaceted roles in tumor metastasis .

How can I implement multiplexed detection systems incorporating ITGB3 antibodies for advanced tissue analysis?

Implementing multiplexed detection systems with ITGB3 antibodies enables comprehensive spatial and functional analysis of integrin biology in complex tissues:

• Multiplex immunofluorescence strategies:

  • Combine ITGB3 antibodies with antibodies against alpha subunits (αV, αIIb) to visualize heterodimer distribution.

  • Use antibodies from different host species (mouse ITGB3/2145 and rabbit ITGB3/2166R) with species-specific secondaries for simultaneous detection of different epitopes .

  • Implement sequential tyramide signal amplification for multiple rounds of staining on the same section.

• Spectral imaging and unmixing:

  • Utilize spectral detectors and unmixing algorithms to separate closely overlapping fluorophores.

  • Combine with autofluorescence removal techniques to enhance signal-to-noise ratios in tissues with high background (e.g., lung, skin).

• Multiplex chromogenic IHC:

  • Apply multiplexed chromogenic detection using antibodies like ITGB3/2145 or ITGB3/2166R with different chromogens for brightfield analysis .

  • Implement machine learning algorithms for automated quantification of staining patterns and co-localization.

• Mass cytometry approaches:

  • Conjugate anti-ITGB3 antibodies with rare earth metals for CyTOF analysis.

  • Combine with imaging mass cytometry for high-dimensional spatial profiling of ITGB3 in relation to dozens of other markers.

• Advanced co-localization techniques:

  • Apply proximity ligation assays (PLA) using anti-ITGB3 antibodies together with antibodies against suspected interaction partners.

  • Implement super-resolution microscopy (STORM, PALM) using directly-labeled ITGB3 antibodies to visualize nanoscale clustering and organization.

• Digital spatial profiling:

  • Incorporate ITGB3 antibodies into digital spatial profiling panels for region-specific quantitative analysis.

  • Correlate with transcriptomic data to integrate protein and RNA expression patterns.

• Considerations for optimization:

  • Carefully test for antibody cross-reactivity in multiplexed settings.

  • Validate staining patterns with individual antibodies before combining.

  • Consider carrier-free antibody formulations to minimize background in multiplexed applications .

These advanced approaches transform standard ITGB3 detection into comprehensive spatial and functional analysis of integrin biology in complex tissues .

How do I validate ITGB3 antibody specificity for my particular experimental system?

Rigorous validation of ITGB3 antibody specificity is essential for reliable experimental results. Implement this comprehensive validation strategy:

• Positive and negative control samples:

  • Use tissues/cells known to express high levels of ITGB3 (platelets, megakaryocytes) as positive controls .

  • Include tissues known to lack ITGB3 expression as negative controls.

  • Test the antibody on species-matched samples, as some antibodies are specifically validated for human or mouse samples .

• Molecular validation approaches:

  • Implement gene silencing (siRNA, shRNA) or genetic knockout (CRISPR/Cas9) of ITGB3 and confirm loss of antibody signal.

  • Perform rescue experiments with exogenous ITGB3 expression to restore antibody reactivity.

  • Use cell lines with characterized ITGB3 expression levels to confirm signal proportionality.

• Multi-antibody concordance:

  • Compare staining patterns using multiple antibodies targeting different ITGB3 epitopes (e.g., ITGB3/2145 and ITGB3/2166R) .

  • Verify concordance between monoclonal and polyclonal antibodies when possible.

• Cross-reactivity assessment:

  • Test on cells overexpressing related integrins (β1, β5, β6) to check for cross-reactivity.

  • Perform peptide competition assays using the immunizing peptide (where available) to confirm epitope specificity.

• Method-specific validations:

  • For Western blot: Confirm appropriate molecular weight (105 kDa / 90 kDa) and band pattern .

  • For IHC/IF: Verify expected subcellular localization (primarily membrane) and tissue distribution .

  • For flow cytometry: Compare with established ITGB3/CD61 antibodies and confirm expected cell population staining.

• Heterodimer specificity:

  • For antibodies claiming heterodimer specificity (like IPI-ITGAV/ITGB3.7), confirm selective binding to cells expressing both subunits versus single subunit expression .

What are the key considerations when selecting between mouse and rabbit monoclonal ITGB3 antibodies?

Selecting between mouse monoclonal [ITGB3/2145] and rabbit monoclonal [ITGB3/2166R] ITGB3 antibodies requires careful consideration of several factors:

• Technical performance differences:

  • Background levels: Rabbit monoclonals often exhibit lower background staining in IHC applications, particularly in mouse tissues (due to endogenous mouse IgG) .

  • Sensitivity: Rabbit monoclonal antibodies frequently demonstrate higher affinity and sensitivity compared to mouse monoclonals due to different antibody generation and selection methods .

  • Epitope recognition: While both antibodies target similar regions (around amino acids 385-490 of ITGB3), subtle differences in epitope recognition may exist .

• Application-specific considerations:

  • Multiplex staining: When performing multiplex immunofluorescence with other antibodies, host species becomes critical. Select mouse ITGB3 antibodies when other primary antibodies are rabbit-derived, and vice versa .

  • Secondary detection: Consider available secondary antibodies and detection systems in your laboratory.

  • Special applications: For certain applications (like proximity ligation assays), having antibodies from different species recognizing the same target is advantageous.

• Formulation differences:

  • Storage buffer: Both antibodies are available with or without BSA and sodium azide. For sensitive applications (cell culture, functional assays), choose azide-free formulations .

  • Concentration: Mouse monoclonal is supplied at 200 μg/ml, while rabbit monoclonal is available at 1 mg/ml, affecting dilution calculations .

• Cross-reactivity profiles:

  • Species reactivity: Verify validated species reactivity - both are validated for human ITGB3, with some showing cross-reactivity to mouse .

  • Isotype considerations: Mouse ITGB3/2145 is IgG2b kappa, while rabbit ITGB3/2166R is IgG, potentially affecting non-specific binding through Fc receptors .

• Practical considerations:

  • Cost and availability: Compare pricing and lead times between the options.

  • Previous laboratory experience: Consider previous success with either antibody type in your laboratory.

Making an informed choice between mouse and rabbit monoclonal antibodies based on these considerations will optimize experimental outcomes and data quality .

What are the critical differences between antibodies targeting ITGB3 alone versus those targeting specific integrin heterodimers?

Understanding the critical differences between antibodies targeting ITGB3 alone versus those targeting specific heterodimeric complexes is essential for experimental design and data interpretation:

• Epitope accessibility and recognition:

  • ITGB3-specific antibodies (like ITGB3/2145 and ITGB3/2166R) recognize epitopes on the beta-3 subunit regardless of its alpha subunit partner, detecting both αIIbβ3 and αVβ3 complexes .

  • Heterodimer-specific antibodies (like IPI-ITGAV/ITGB3.7) recognize conformational epitopes formed by the interface between specific alpha and beta subunits, selectively binding only to particular heterodimers (e.g., αVβ3 but not αIIbβ3) .

• Functional implications:

  • ITGB3-specific antibodies are ideal for total ITGB3 expression studies but may not distinguish between functional heterodimers.

  • Heterodimer-specific antibodies provide precise information about specific integrin complexes and can often block ligand binding to specific heterodimers without affecting other ITGB3-containing complexes .

• Application advantages:

  • Tissue distribution studies: ITGB3-specific antibodies provide comprehensive detection of all ITGB3-containing complexes .

  • Functional studies: Heterodimer-specific antibodies with ligand-blocking properties (like the RGD-mimetic IPI-ITGAV/ITGB3.7) can selectively inhibit specific integrin functions .

  • Cancer research: Heterodimer-specific antibodies can distinguish between platelet-associated αIIbβ3 and tumor-associated αVβ3, critical for interpreting tumor microenvironment studies.

• Technical performance differences:

  • Signal intensity: ITGB3-specific antibodies may yield stronger signals in tissues expressing multiple ITGB3-containing heterodimers.

  • Specificity challenges: Heterodimer-specific antibodies may have more stringent validation requirements to confirm selective binding.

• Experimental design considerations:

  • Use heterodimer-specific antibodies when studying specific functions (e.g., αVβ3 in angiogenesis or tumor invasion).

  • Use ITGB3-specific antibodies for general expression profiling or when comprehensive detection is desired.

  • Consider using both types in parallel for complete characterization of ITGB3 biology.