CD31 Biotin Antibody

What is CD31 and why is it a significant target for biotin-conjugated antibodies?

CD31, also known as platelet-endothelial cell adhesion molecule-1 (PECAM-1) and gpIIa, is a 130-140 kDa transmembrane glycoprotein expressed primarily by endothelial cells and at lower levels on leukocytes and platelets . As a member of the immunoglobulin supergene family of adhesion molecules, CD31 plays critical roles in vascular biology through its homotypic binding interactions that mediate cell-cell adhesion, particularly at endothelial junctions . The strategic biotinylation of anti-CD31 antibodies enhances detection sensitivity through the strong biotin-streptavidin interaction, providing researchers with powerful tools for visualizing and quantifying CD31 expression in tissues and cells with exceptional specificity and signal amplification capabilities .

How do different CD31 Biotin Antibody clones compare in research applications?

Several key monoclonal antibody clones have been developed against CD31, each with distinct characteristics that make them suitable for specific research applications:

When selecting the appropriate clone, researchers should consider the specific experimental needs, target species, fixation methods, and downstream applications. For detecting low-abundance CD31 expression, higher-affinity clones like EPR3094 may provide superior results, while WM-59 is widely validated for flow cytometry applications .

What are the optimal protocols for using CD31 Biotin Antibody in flow cytometric analysis?

For optimal flow cytometric analysis with CD31 Biotin antibodies, the following methodological approach is recommended:

• Sample preparation: Prepare single-cell suspensions from your tissue of interest (e.g., peripheral blood, spleen, or cultured endothelial cells).

• Cell count adjustment: Determine cell concentration empirically, with a typical range of 10^5 to 10^8 cells per test in a final volume of 100 μL .

• Antibody titration: Critical for optimal signal-to-noise ratio. Start with ≤0.25 μg per test for clones like WM-59 or TLD-3A12, but perform a titration series (e.g., 0.05, 0.1, 0.25, 0.5 μg) to determine the optimal concentration for your specific experimental conditions .

• Staining procedure:

  • Block non-specific binding with appropriate serum

  • Incubate cells with biotin-conjugated CD31 antibody at 4°C for 30 minutes

  • Wash cells twice with flow cytometry buffer

  • Incubate with streptavidin-fluorophore conjugate (e.g., streptavidin-PE)

  • Wash twice before analysis

• Controls:

  • Include isotype controls matched to your primary antibody

  • Include unstained cells and single-stained controls for compensation

  • For multicolor panels, include fluorescence-minus-one (FMO) controls

• Analysis parameters: Gate on relevant populations based on forward/side scatter characteristics before analyzing CD31 expression. For endothelial cells, consider co-staining with other endothelial markers such as CD34 or VEGFR2 .

How can researchers effectively use CD31 Biotin Antibody to quantify tumor angiogenesis?

CD31 Biotin Antibody serves as a powerful tool for quantifying tumor angiogenesis through the following methodological approach:

• Tissue processing:

  • Fix tumor tissues in 10% neutral buffered formalin

  • Process and embed in paraffin

  • Section at 4-5 μm thickness

• Antigen retrieval:

  • Perform heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Optimize retrieval time (typically 15-20 minutes)

• Staining protocol:

  • Block endogenous peroxidase and biotin

  • Apply biotin-conjugated CD31 antibody (e.g., clone C31.3) at optimized concentration

  • Incubate at 4°C overnight or room temperature for 1-2 hours

  • Detect using streptavidin-HRP and appropriate chromogen (e.g., DAB)

• Quantification methods:

  • Microvessel density (MVD): Count CD31-positive vessels in "hot spots" (areas of highest vessel density) at 200-400× magnification

  • Total vessel area: Measure total CD31-positive area using image analysis software

  • Vessel morphology: Quantify vessel diameter, branching, and pattern

• Interpretation:

  • High levels of CD31 expression may indicate rapidly growing tumors and potentially predict tumor recurrence

  • Compare MVD values across different tumor regions and patient samples

  • Correlate with clinical parameters and patient outcomes

This approach provides objective quantification of tumor angiogenesis, enabling researchers to assess vascular patterns and potential therapeutic targets in cancer research .

How does CD31 facilitate leukocyte transendothelial migration, and how can this be studied using CD31 Biotin Antibody?

CD31 plays a critical role in leukocyte transendothelial migration (TEM) through complex molecular mechanisms that can be effectively studied using CD31 Biotin Antibody:

Molecular mechanisms of CD31-mediated TEM:

• CD31 is located primarily at endothelial cell junctions and functions as a molecular gate for transmigrating leukocytes .

• Tyrosine-690 plays a critical role in TEM by regulating the trafficking of CD31 to and from the lateral border recycling compartment (LBRC) .

• The LBRC membrane must be targeted around migrating leukocytes, with Tyr-690 being essential for this process .

• CD31 mediates both homophilic (CD31-CD31) interactions and heterophilic interactions, such as with CD177 on neutrophils, facilitating their transendothelial migration .

Experimental approaches using CD31 Biotin Antibody:

• In vitro transmigration assays:

  • Grow endothelial cell monolayers on permeable supports

  • Add fluorescently labeled leukocytes to the upper chamber

  • Block CD31 function using CD31 Biotin Antibody

  • Quantify transmigration by counting leukocytes in the lower chamber

• Live cell imaging:

  • Visualize the redistribution of CD31 during TEM by staining with low concentrations of CD31 Biotin Antibody

  • Track the recruitment of the LBRC around transmigrating leukocytes

  • Monitor CD31 phosphorylation state during TEM using phospho-specific antibodies

• Site-directed mutagenesis studies:

  • Create mutants of CD31 (e.g., Y690F) to study the role of specific residues

  • Use CD31 Biotin Antibody to detect expression and localization of mutant proteins

  • Analyze the impact on TEM efficiency and LBRC trafficking

• In vivo studies:

  • Use CD31 Biotin Antibody to track endothelial junction integrity during inflammation

  • Combine with intravital microscopy to visualize leukocyte-endothelial interactions in real-time

By employing these approaches, researchers can dissect the complex mechanisms by which CD31 regulates leukocyte transendothelial migration under inflammatory conditions .

What role does CD31 play in apoptosis regulation and phagocytosis, and how can CD31 Biotin Antibody be used to investigate these processes?

CD31 exhibits dual functionality in apoptosis regulation and phagocytosis that can be comprehensively investigated using CD31 Biotin Antibody:

CD31's role in apoptosis and phagocytosis:

• CD31 homophilic ligation prevents macrophage-mediated phagocytosis of viable leukocytes by transmitting a "detachment signal" .

• This detachment signal appears to be disabled in apoptotic leukocytes, allowing CD31 to promote macrophage-mediated phagocytosis of these cells .

• CD31 tethers apoptotic leukocytes to phagocytic cells, facilitating clearance .

• Isoform Delta15 of CD31 does not protect against apoptosis, suggesting isoform-specific functions .

Methodological approaches using CD31 Biotin Antibody:

• Apoptosis assays:

  • Induce apoptosis in leukocytes using appropriate stimuli (e.g., UV radiation, FasL)

  • Use CD31 Biotin Antibody to monitor CD31 expression, distribution, and modification during apoptosis

  • Combine with annexin V staining to correlate CD31 status with apoptotic stages

• Phagocytosis assays:

  • Label target cells (viable or apoptotic) with fluorescent dyes

  • Incubate with macrophages in the presence or absence of CD31-blocking antibodies

  • Quantify phagocytosis by flow cytometry or confocal microscopy

  • Use CD31 Biotin Antibody to visualize CD31 redistribution during phagocytic events

• Isoform-specific analysis:

  • Use RT-PCR to determine expression of different CD31 isoforms in your cellular system

  • Develop isoform-specific detection methods using CD31 Biotin Antibody

  • Compare functional outcomes (apoptosis protection, phagocytosis) between isoforms

• Signaling pathway investigation:

  • Use CD31 Biotin Antibody to precipitate CD31 and associated proteins

  • Analyze phosphorylation status of immunoreceptor tyrosine-based inhibitory motifs (ITIMs)

  • Investigate recruitment of protein-tyrosine phosphatases to CD31

These methodological approaches allow researchers to dissect the complex and sometimes contradictory roles of CD31 in regulating cell survival and clearance mechanisms .

How should researchers address inconsistent CD31 Biotin Antibody staining results across different experimental systems?

• Antibody-related factors:

  • Clone specificity: Different clones recognize distinct epitopes that may be differentially accessible in various experimental systems. For human samples, compare WM-59 versus C31.3 or EPR3094 performance .

  • Antibody concentration: Perform titration experiments to determine optimal concentration. Typical starting points are ≤0.25 μg per test for flow cytometry .

  • Biotin:antibody ratio: Over-biotinylation can reduce antibody affinity. Verify biotinylation level with the manufacturer.

  • Antibody age and storage: Biotin-conjugated antibodies may deteriorate over time. Store at manufacturer-recommended temperatures and avoid repeated freeze-thaw cycles.

• Sample-related factors:

  • Fixation effects: CD31 epitopes can be masked by certain fixatives. Compare fresh versus fixed samples and optimize fixation time.

  • Antigen retrieval methods: For FFPE tissues, compare citrate (pH 6.0) versus EDTA (pH 9.0) buffers and adjust retrieval times.

  • Tissue-specific expression levels: CD31 is highly expressed on endothelial cells but at lower levels on leukocytes and platelets . Adjust exposure/gain settings accordingly.

  • Species differences: Ensure the antibody clone is validated for your species (e.g., TLD-3A12 for rat, WM-59 for human) .

• Protocol optimization:

  • Blocking steps: Increase blocking time/concentration to reduce non-specific binding.

  • Incubation conditions: Compare room temperature versus 4°C incubation.

  • Washing stringency: Adjust washing buffer composition and number of washes.

  • Detection system: For biotin-streptavidin systems, block endogenous biotin and compare different streptavidin conjugates.

• Validation approaches:

  • Multiple antibody approach: Use two different CD31 antibody clones targeting different epitopes.

  • Complementary methods: Validate findings with alternative techniques (e.g., flow cytometry, Western blot, IF, IHC).

  • Positive and negative controls: Always include known positive and negative tissues/cells.

  • Genetic validation: Use CD31 knockout or knockdown systems when available.

By systematically addressing these factors, researchers can resolve inconsistencies and generate reliable, reproducible CD31 staining data across different experimental systems .

What are the best practices for quantifying CD31 expression in tumor samples using CD31 Biotin Antibody?

Accurate quantification of CD31 expression in tumor samples requires standardized methodologies:

• Sampling considerations:

  • Analyze multiple tumor regions to account for intratumoral heterogeneity

  • Include tumor margins and center to capture spatial differences in vascularization

  • Use standardized tissue size and thickness (typically 4-5 μm for FFPE sections)

• Staining protocol standardization:

  • Optimize and standardize antigen retrieval conditions

  • Use automated staining platforms when possible to ensure consistency

  • Include positive control tissues (e.g., tonsil) in each staining batch

  • Process all comparative samples in the same staining run

• Quantification methods and metrics:

  Metric	Method	Advantages	Limitations
  Microvessel Density (MVD)	Count CD31+ vessels in hotspots (typically 3-5 fields at 200× magnification)	Widely used, established prognostic value	Observer-dependent, neglects vessel size/function
  Vessel Area	Measure total CD31+ area using image analysis software	Less subjective, accounts for vessel size	Requires standardized image acquisition
  Vessel Architecture	Assess branching, diameter, shape factors	Provides information on vessel maturity/function	Complex analysis, requires specialized software
  Vascular Gradient	Measure CD31 expression from tumor center to periphery	Captures spatial heterogeneity	Labor-intensive, requires larger samples

• Digital image analysis recommendations:

  • Use calibrated image acquisition with standardized exposure settings

  • Implement validated segmentation algorithms for vessel detection

  • Apply consistent thresholding methods across all samples

  • Report multiple metrics (e.g., MVD, vessel area, vessel diameter)

• Data interpretation guidelines:

  • High levels of CD31 expression may indicate rapidly growing tumors and potentially predict tumor recurrence

  • Compare MVD values to established benchmarks for specific tumor types

  • Consider CD31 expression alongside other angiogenesis markers (e.g., VEGF)

  • Correlate with clinical parameters and survival data when available

By following these standardized approaches, researchers can generate robust, reproducible quantification of tumor vasculature that facilitates meaningful comparisons across studies and potential translation to clinical applications .

How is CD31 Biotin Antibody being utilized in single-cell analysis technologies for vascular and immune cell research?

The integration of CD31 Biotin Antibody with cutting-edge single-cell technologies is revolutionizing vascular and immune cell research:

• Single-cell RNA sequencing (scRNA-seq) applications:

  • CD31 Biotin Antibody can be used for initial cell isolation via magnetic separation or FACS

  • This enables enrichment of rare endothelial subpopulations for subsequent scRNA-seq analysis

  • Researchers can correlate CD31 protein expression levels with transcriptomic profiles

  • This approach has revealed previously unrecognized endothelial heterogeneity across different vascular beds

• CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing):

  • CD31 Biotin Antibody can be conjugated to oligonucleotide barcodes

  • This allows simultaneous measurement of CD31 protein expression and transcriptome in the same cell

  • Researchers can identify heterogeneous functional states within CD31+ populations

  • The approach reveals relationships between CD31 expression levels and specific transcriptional programs

• Spatial transcriptomics integration:

  • CD31 Biotin Antibody staining can be combined with spatial transcriptomics methods

  • This preserves spatial information about CD31+ vessels in tissue contexts

  • Researchers can analyze gene expression changes in proximity to CD31+ vessels

  • This approach is particularly valuable for studying tumor microenvironments and angiogenesis

• Mass cytometry (CyTOF) applications:

  • CD31 Biotin Antibody can be used with metal-tagged streptavidin for CyTOF analysis

  • This enables high-dimensional phenotyping of CD31+ cells

  • Researchers can simultaneously examine dozens of other markers alongside CD31

  • This approach has revealed complex phenotypic signatures of endothelial cells in different physiological and pathological states

• Microfluidic approaches:

  • CD31 Biotin Antibody-coated microchannels can capture circulating endothelial cells

  • This enables downstream single-cell analysis of rare vascular cells in blood

  • Researchers can study endothelial dysfunction in cardiovascular diseases

  • The approach facilitates development of liquid biopsy applications

These emerging single-cell applications of CD31 Biotin Antibody are providing unprecedented insights into endothelial cell biology, leukocyte-endothelial interactions, and vascular heterogeneity in both normal physiology and disease states .

What are the latest findings on CD31's role in immune regulation and how can CD31 Biotin Antibody advance this research?

Recent research has revealed CD31's sophisticated functions in immune regulation, which can be further explored using CD31 Biotin Antibody:

• CD31 as an inhibitory immune receptor:

  • CD31 contains dual immunoreceptor tyrosine-based inhibitory motifs (ITIMs) that regulate T cell and B cell signaling

  • Upon phosphorylation by associated kinases, these ITIMs provide docking sites for protein-tyrosine phosphatases

  • CD31 Biotin Antibody can be used to track ITIM phosphorylation status in different immune contexts

  • Researchers can investigate how CD31 signaling modulates immune cell activation thresholds

• CD31 in autoimmunity regulation:

  • Recent studies implicate CD31 in preventing excessive immune responses that could lead to autoimmunity

  • CD31 Biotin Antibody can be used to phenotype CD31 expression patterns in autoimmune disease models

  • Researchers can correlate CD31 expression levels with disease severity and progression

  • This enables investigation of CD31 as a potential therapeutic target for autoimmune conditions

• Role in allergic responses:

  • CD31 has been implicated in regulating IgE-mediated anaphylaxis

  • CD31 Biotin Antibody can be used to study CD31 dynamics during mast cell degranulation

  • Researchers can investigate how CD31 signaling interfaces with FcεRI pathways

  • This research direction may lead to novel approaches for allergic disease management

• CD31 in immunological synapse formation:

  • Emerging evidence suggests CD31 may regulate immunological synapse stability

  • CD31 Biotin Antibody enables high-resolution imaging of CD31 localization during immune cell interactions

  • Researchers can use super-resolution microscopy with CD31 Biotin Antibody to study nanoscale organization

  • This approach reveals how CD31 positioning influences signaling outcomes in immune cells

• CD31 in immune cell trafficking:

  • Beyond its role in transendothelial migration, CD31 influences immune cell positioning within tissues

  • CD31 Biotin Antibody can be used in intravital imaging studies to track cell migration dynamics

  • Researchers can examine how CD31 regulates immune cell retention in inflammatory sites

  • This research enhances understanding of immune surveillance and inflammatory resolution

By leveraging CD31 Biotin Antibody in these emerging research areas, investigators can gain deeper insights into CD31's multifaceted roles in immune regulation, potentially leading to novel therapeutic strategies for inflammatory, autoimmune, and allergic disorders .