CD31 Antibody

What is CD31 and what is its significance in cellular biology?

CD31, also known as Platelet Endothelial Cell Adhesion Molecule-1 (PECAM-1) or Endocam, is a 130-140 kD type I transmembrane glycoprotein expressed on endothelial cells and in platelets . It belongs to the immunoglobulin gene superfamily and is primarily expressed at high density at the lateral borders of endothelial cells and at lower density on various hematopoietic cells including T lymphocytes . CD31 serves multiple critical functions in cellular biology, including establishing homophilic interactions between adjacent endothelial cells, facilitating endothelium-leukocyte interactions, and contributing to endothelial cell-cell junctions . Additionally, it plays significant roles in cell signaling and cell adhesion mechanisms, which are fundamental to vascular integrity and immune response coordination . Research has demonstrated that CD31 functions as a nonredundant comodulator of T-cell responses by setting thresholds for T-cell activation and tolerance .

Which cell types express CD31 and how does expression level vary?

CD31 exhibits a diverse expression pattern across multiple cell types, making it a valuable marker for identifying and studying various cell populations. According to antibody data sheets, CD31 is expressed on:

• Endothelial cells (highest expression, particularly at cell junctions)

• Fibroblasts

• Various immune cells including granulocytes, macrophages, monocytes, NK cells

• Osteoclasts

• Platelets

• T cells

• Basophils

• Neutrophils

• Plasma cells

Expression levels vary significantly between cell types, with endothelial cells showing the most robust expression. This differential expression pattern is important to consider when designing flow cytometry panels or interpreting immunohistochemistry results, as detection sensitivity may need to be optimized differently depending on the target cell population. The high expression on endothelial cells makes CD31 a particularly useful marker for vascular research and angiogenesis studies.

What are the molecular characteristics of CD31 relevant to antibody selection?

Understanding CD31's molecular characteristics is crucial for appropriate antibody selection. CD31 has a molecular mass of approximately 79 kDa as the core protein , but typically appears at 130-165 kDa in Western blots due to extensive glycosylation . This significant difference between predicted and observed molecular weight is an important consideration when validating antibody specificity by Western blot.

Structurally, CD31 is a type I transmembrane protein containing six extracellular immunoglobulin-like domains, a transmembrane region, and a cytoplasmic tail with signaling motifs. Different antibody clones recognize distinct epitopes on CD31, which can affect their utility in specific applications. For example, clone WM59 recognizes the D2 extracellular portion of CD31 , making it suitable for applications where this domain is accessible.

When selecting CD31 antibodies, researchers should consider:

• The specific epitope recognized and whether it might be affected by fixation or processing

• Species reactivity (e.g., human-specific, mouse-specific, or cross-reactive antibodies)

• Clone performance in specific applications (some clones work well for flow cytometry but poorly for immunohistochemistry)

• The isotype of the antibody for appropriate control selection (e.g., rat IgG2aκ for clone 390)

How should samples be prepared for optimal CD31 immunohistochemistry?

Successful CD31 immunohistochemistry requires careful attention to sample preparation. Based on product documentation, the following protocol is recommended:

For paraffin-embedded tissues:

• Deparaffinize and rehydrate tissue sections following standard protocols

• Perform heat-mediated antigen retrieval using 10mM citrate buffer (pH 6.0) for 10 minutes, followed by cooling at room temperature for 20 minutes

• Block endogenous peroxidase activity (if using HRP detection)

• Apply protein block to reduce non-specific binding

• Incubate with primary CD31 antibody at an optimized dilution (1:50 is recommended for some antibodies)

• Incubate for 30 minutes at room temperature

• Wash thoroughly with buffer

• Apply appropriate detection system

• Counterstain, dehydrate, and mount

For frozen sections:

• Note that some clones (e.g., WM59) are specifically recommended for acetone-fixed frozen sections but not for formalin-fixed paraffin-embedded sections

• Fix sections in cold acetone for 10 minutes

• Allow sections to air dry

• Rehydrate in buffer

• Follow steps 3-9 as above

Critical considerations include:

• Tonsil tissue serves as an excellent positive control for human CD31 staining

• Antibody dilution may require optimization for different tissue types

• Some epitopes are particularly sensitive to fixation method and duration

What are the optimal protocols for CD31 detection by flow cytometry?

For reliable flow cytometric analysis of CD31 expression, the following protocol is recommended based on manufacturer guidelines:

• Prepare a single-cell suspension:

  • Use enzymatic digestion methods that preserve surface epitopes

  • For tissues containing endothelial cells, gentle digestion is crucial

  • Adjust cell concentration to approximately 1 × 10^6 cells per 100 μL

• Staining procedure:

  • Wash cells in flow cytometry buffer (PBS with 0.5-1% BSA and 2mM EDTA)

  • Block Fc receptors if needed to reduce non-specific binding

  • Add CD31 antibody at appropriate dilution (1:50 is recommended for some antibodies)

  • Incubate for 20-30 minutes at 2-8°C protected from light

  • Wash cells twice to remove unbound antibody

  • If using biotin-conjugated primary antibody, add fluorochrome-conjugated streptavidin secondary

  • Resuspend in appropriate buffer for analysis

• Critical considerations:

  • Cells should be stained prior to fixation if formaldehyde is used as a fixative

  • When designing multicolor panels, consider that CD31 is commonly detected with PE, which is excited by Green (532 nm) or Yellow-Green (561 nm) lasers

  • Include appropriate isotype controls (e.g., rat IgG2aκ for clone 390)

  • For endothelial cells, additional markers like VE-cadherin may help distinguish different vascular cell populations

• Data analysis recommendations:

  • Use forward and side scatter to identify intact cells

  • Apply consistent gating strategies between experiments

  • Consider CD31 expression intensity when interpreting results (endothelial cells express higher levels than leukocytes)

What are the critical factors for successful Western blot detection of CD31?

Western blot detection of CD31 requires specific considerations due to its high molecular weight and glycosylation status. Based on product documentation, the following factors are critical:

• Sample preparation:

  • Use lysis buffers that effectively solubilize membrane proteins

  • Include protease inhibitors to prevent degradation

  • For positive controls, consider endothelial cell lines like bEnd.3 mouse endothelioma cells

• Gel electrophoresis parameters:

  • Use lower percentage gels (7-10%) to effectively resolve the high molecular weight CD31 protein

  • Run samples under reducing conditions for optimal detection

  • Load sufficient protein (20-50 μg of total protein per lane)

• Transfer considerations:

  • Employ longer transfer times for high molecular weight proteins

  • Use PVDF membrane for better protein retention

  • Consider wet transfer methods for large proteins

• Detection parameters:

  • Anticipate CD31 to appear at approximately 130-165 kDa due to glycosylation

  • Use antibodies specifically validated for Western blot (e.g., AF3628 at 0.5 μg/mL concentration)

  • Block with appropriate blocking reagents (often BSA-based)

  • Use specific immunoblot buffer groups as recommended (e.g., Immunoblot Buffer Group 1)

• Troubleshooting common issues:

  • If no band is detected, consider increasing protein loading or antibody concentration

  • Multiple bands may represent different glycosylation states or degradation products

  • High background may require more stringent washing or decreased antibody concentration

Example data from product documentation shows successful detection of mouse CD31 in bEnd.3 cell lysates, with a specific band at approximately 130 kDa under reducing conditions .

How can researchers optimize CD31 antibody titration for different applications?

Optimizing CD31 antibody concentration is essential for balancing specific signal with background. A systematic approach to antibody titration includes:

• Flow cytometry titration:

  • Begin with manufacturer's recommended dilution (often 1:50)

  • Prepare a series of dilutions (e.g., 1:25, 1:50, 1:100, 1:200)

  • Stain identical aliquots of a positive control sample with each dilution

  • Analyze signal-to-noise ratio by comparing median fluorescence intensity (MFI) of positive and negative populations

  • Select the dilution that provides maximum separation with minimal background

  • For reproducible results, express the optimal concentration in μg/mL rather than dilution factor

• Immunohistochemistry optimization:

  • Start with recommended dilution (e.g., 1:50 for some antibodies)

  • Prepare a dilution series (e.g., 1:25, 1:50, 1:100, 1:200)

  • Use consistent positive control tissue (tonsil is recommended for human CD31)

  • Evaluate staining intensity, specificity, and background at each dilution

  • Consider antigen retrieval variations if staining is suboptimal

  • Document optimal conditions for future reference

• Western blot optimization:

  • Begin with recommended concentration (e.g., 0.5 μg/mL for some antibodies)

  • Test a range of concentrations while keeping all other variables constant

  • Include positive control lysates (e.g., endothelial cell lines)

  • Evaluate band specificity, intensity, and background

  • Remember that detection method sensitivity (chemiluminescence vs. fluorescence) may necessitate different antibody concentrations

• Documentation of optimization:

  • Create a table documenting optimal conditions for each application

  • Record batch/lot numbers of antibodies, as optimal concentration may vary between lots

  • Include sample types tested and detection methods used

What controls are essential when using CD31 antibodies in multi-parameter analysis?

Multi-parameter analysis using CD31 antibodies requires rigorous controls to ensure reliable results:

• Essential controls for flow cytometry:

  • Isotype control matched to CD31 antibody (e.g., rat IgG2aκ for clone 390)

  • Fluorescence minus one (FMO) controls to set accurate gates

  • Single-stained controls for compensation when using multiple fluorophores

  • Positive control samples (e.g., endothelial cells)

  • Negative control samples (cell types known not to express CD31)

  • Viability dye to exclude dead cells (which can bind antibodies non-specifically)

• Controls for immunohistochemistry/immunofluorescence:

  • Isotype-matched primary antibody control at the same concentration

  • Secondary antibody-only control to assess non-specific binding

  • Known positive tissue control (tonsil recommended for human CD31)

  • Negative tissue control (tissue known not to express CD31)

  • Autofluorescence control for immunofluorescence studies

• Western blot controls:

  • Positive control lysate (e.g., endothelial cell line)

  • Loading control (e.g., β-actin, GAPDH)

  • Molecular weight markers to confirm the expected band size (130-165 kDa for CD31)

  • Non-expressing cell line as negative control

• Advanced multiplex controls:

  • For IBEX and other multiplexed imaging techniques, additional controls to assess antibody stripping efficiency and epitope stability through multiple cycles

  • Cross-reactivity controls when using multiple primary antibodies of the same species

  • Spectral overlap controls for multispectral imaging systems

How can researchers troubleshoot inconsistent CD31 staining patterns?

Inconsistent CD31 staining patterns are a common challenge that can be systematically addressed:

• Sample preparation variables:

  • Fixation effects: Some CD31 epitopes are fixation-sensitive; clone WM59 works well with acetone-fixed frozen sections but not with formalin-fixed paraffin-embedded sections

  • Antigen retrieval efficacy: Heat-mediated antigen retrieval using citrate buffer (pH 6.0) significantly impacts staining for some antibodies

  • Processing time: Delays between sample collection and fixation can reduce antigen integrity

• Technical troubleshooting strategies:

  • For weak staining:

    • Increase antibody concentration

    • Optimize antigen retrieval (adjust time, buffer, or temperature)

    • Extend primary antibody incubation time

    • Use more sensitive detection systems

  • For high background:

    • Increase blocking (duration or concentration)

    • Dilute primary antibody

    • Add detergents to wash buffers

    • Reduce incubation temperatures

• Antibody-specific considerations:

  • Different clones recognize different epitopes; clone WM59 recognizes the D2 extracellular portion

  • Some antibodies perform inconsistently after freeze-thaw cycles

  • Storage conditions impact stability (store at 2-8°C protected from light)

  • Consider lot-to-lot variations and validate new lots against previously successful experiments

• Biological variation considerations:

  • CD31 expression levels vary by cell type (high on endothelial cells, lower on leukocytes)

  • Expression patterns differ (concentrated at endothelial cell junctions versus diffuse membrane staining on leukocytes)

  • Pathological conditions may alter expression patterns

• Systematic validation approach:

  • Use multiple techniques to confirm observations (e.g., flow cytometry, Western blot, and IHC)

  • Compare results with literature reports

  • Test multiple antibody clones if inconsistencies persist

Implementing this structured troubleshooting approach can help researchers achieve consistent, reproducible CD31 staining across different experimental conditions and sample types.

How can CD31 antibodies be utilized to study T-cell immunomodulation?

CD31 antibodies offer powerful tools for investigating its nonredundant role in T-cell responses:

• Experimental approaches to study CD31's effect on T-cell activation:

  • Use purified CD31 antibodies to modulate CD31 signaling in T-cell cultures

  • Assess effects on proximal TCR signaling, specifically Zap-70 phosphorylation, which is partly inhibited by CD31

  • Compare wildtype versus CD31-deficient T-cells for differences in activation threshold and tolerance induction

  • Analyze clonal expansion dynamics in the presence or absence of CD31 signaling

• Methods to investigate CD31's role in T-cell survival:

  • Utilize CD31 antibodies to trigger Erk-mediated prosurvival activity in T cells

  • Study this pathway both in conjunction with TCR signaling and autonomously

  • Measure activation-induced cell death in CD31-sufficient versus CD31-deficient T cells

  • Analyze memory T-cell survival when CD31 signaling is present or blocked

• Functional assays to assess CD31's impact on regulatory T-cell function:

  • Compare suppressive capacity of wildtype versus CD31-deficient Tregs

  • Use Ultra-LEAF™ purified antibodies (endotoxin < 0.01 EU/μg, azide-free) for functional assays

  • Measure changes in regulatory markers and cytokine production

• In vivo applications:

  • Tumor rejection models to assess impact of CD31 manipulation

  • Allograft rejection studies

  • Tolerance induction protocols

  • Autoimmunity models

These approaches leverage the research finding that CD31 functions as a comodulator that "sizes" the ensuing immune response by setting the threshold for T-cell activation and tolerance, while preventing memory T-cell death . This makes CD31 a fascinating target for both basic immunology research and potential therapeutic interventions.

What methodologies exist for using CD31 antibodies in spatial biology and multiplex imaging?

Advanced spatial biology applications represent cutting-edge uses of CD31 antibodies:

• Iterative Bleaching Extended multi-pleXity (IBEX) technology:

  • CD31 antibodies have been validated for IBEX applications

  • This fluorescent imaging technique enables highly-multiplexed spatial analysis

  • The method employs cyclical bleaching of panels of fluorescent antibodies

  • Allows imaging and analysis of many markers over multiple cycles of staining, imaging, and bleaching

  • Enables visualization of CD31+ structures in relationship to numerous other markers simultaneously

• Implementation considerations for spatial biology:

  • Antibody selection: Some clones are specifically validated for spatial applications (e.g., clone WM59)

  • Optimization of antibody concentration for multiplexed detection

  • Validation of epitope stability through multiple rounds of staining and bleaching

  • Determination of optimal imaging parameters for consistent detection

• Analytical approaches:

  • Quantification of spatial relationships between CD31+ structures and other cell types

  • Correlation of vascular patterns with tissue microenvironments

  • Computational analysis of vessel morphology and distribution

  • Integration of spatial data with other experimental modalities

• Applications in disease research:

  • Tumor microenvironment analysis (vascular network in relation to immune infiltrates)

  • Inflammatory disease tissue architecture assessment

  • Developmental biology studies of vascular patterning

  • Regenerative medicine applications

This approach represents a significant advance beyond traditional single or dual-color immunostaining, allowing researchers to understand CD31-expressing structures in their full biological context with dozens of other markers simultaneously, generating rich datasets for computational analysis.

How can CD31 antibodies be employed to investigate endothelial-to-mesenchymal transition?

CD31 antibodies provide valuable tools for studying endothelial-to-mesenchymal transition (EndMT), a process relevant to fibrosis, cardiovascular development, and cancer progression:

• Experimental design for EndMT studies:

  • Use CD31 antibodies to track endothelial phenotype loss during transition

  • Combine with mesenchymal markers (e.g., SM22α, FSP1, α-SMA) to create comprehensive transition panels

  • Monitor CD31 expression changes in response to EndMT inducers like TGF-β2

  • Assess how interventions like AcSDKP affect CD31 expression during EndMT

• Technical approaches:

  • Western blot analysis to quantify CD31 protein levels during transition

  • Flow cytometry to track changing proportions of CD31+ and mesenchymal marker+ populations

  • Immunofluorescence to visualize morphological changes alongside marker shifts

  • Time-course experiments to capture transition dynamics

• Pathway analysis integration:

  • Combine CD31 antibodies with signaling pathway analysis (e.g., p-smad3/smad3 ratio)

  • Investigate cross-talk between TGF-β signaling and FGFR1/FRS2 pathway using CD31 as an endothelial marker

  • Study effects of pathway modulation on CD31 expression and EndMT progression

• Quantitative assessment methods:

  • Densitometric analysis of CD31 Western blots to quantify EndMT progression

  • Flow cytometric quantification of CD31+ cell percentage

  • Image analysis algorithms to measure CD31 staining intensity and distribution

  • Statistical correlations between CD31 levels and mesenchymal marker expression

Research results indicate that interventions like AcSDKP can suppress TGF-β/smad signaling and EndMT through the FGFR1/FRS2 pathway, with effects visible through CD31 expression analysis . These experimental approaches allow researchers to investigate the molecular mechanisms controlling EndMT and potential therapeutic interventions.

What experimental designs best utilize CD31 antibodies for studying vascular-immune interactions?

CD31 antibodies offer unique advantages for investigating vascular-immune interactions due to CD31's expression on both endothelial cells and leukocytes:

• In vitro models of transendothelial migration:

  • Use CD31 antibodies to visualize endothelial junctions where leukocyte migration occurs

  • Function-blocking studies with Ultra-LEAF™ purified antibodies (endotoxin < 0.01 EU/μg, azide-free)

  • Live-cell imaging of CD31-mediated homophilic interactions during leukocyte migration

  • Quantification of migration efficiency with and without CD31 blockade

• Flow cytometry approaches:

  • Multi-parameter analysis to simultaneously identify CD31+ endothelial cells and CD31+ leukocyte populations

  • Sorting of CD31high versus CD31low T cells to investigate functional differences

  • Analysis of how activation status affects CD31 expression on different immune cells

  • Correlation of CD31 expression with functional markers

• Tissue-based analysis:

  • IBEX and other multiplexed imaging techniques to visualize CD31+ vasculature in relation to immune cell infiltrates

  • Quantification of spatial relationships between vasculature and immune cells

  • Assessment of CD31 expression changes during inflammation

  • Analysis of perivascular immune cell clustering

• Signaling studies:

  • Investigation of CD31's role in modulating TCR signaling at the vascular interface

  • Analysis of how CD31-mediated interactions affect both endothelial and immune cell signaling

  • Study of how CD31 signaling influences immune cell recruitment and retention

• Disease model applications:

  • Tumor microenvironment studies (angiogenesis in relation to immune infiltration)

  • Inflammatory disease models (vascular changes during inflammation)

  • Transplant rejection models (as CD31-deficient mice show enhanced allograft rejection)

  • Autoimmune disease models (examining vascular-immune interfaces)

These experimental approaches leverage CD31's unique expression pattern and homophilic binding properties to provide insights into the molecular mechanisms governing vascular-immune interactions in health and disease.

What future directions are emerging for CD31 antibody applications in research?

Recent advances in CD31 antibody applications point to several promising future research directions:

• Advanced spatial biology applications represent a significant frontier, with CD31 antibodies already validated for cutting-edge IBEX technology . This enables highly multiplexed analysis of CD31+ structures in their native tissue context, opening possibilities for detailed architectural analysis of vascular networks in development and disease.

• The nonredundant role of CD31 in T-cell immunomodulation provides opportunities for investigating novel immune checkpoint mechanisms . This could potentially inform new immunotherapeutic approaches, particularly in cancer and transplantation settings where CD31-deficient mice show enhanced tumor and allograft rejection.

• Endothelial-to-mesenchymal transition research using CD31 as a key marker is revealing complex pathway interactions, particularly between TGF-β signaling and the FGFR1/FRS2 pathway . This research direction may yield insights into fibrosis, wound healing, and cardiovascular development.

• Vascular-immune interaction studies leveraging CD31's expression on both vascular and immune cells show promise for understanding recruitment and retention mechanisms in inflammation. The homophilic binding properties of CD31 make it uniquely suited as both a marker and functional target in these studies.

• The continued development of more sensitive and specific CD31 antibodies with diverse conjugates will enable increasingly sophisticated multi-parameter analysis, facilitating deeper understanding of CD31's roles in normal physiology and disease processes.

These emerging directions highlight the continuing importance of CD31 antibodies as versatile tools for investigating fundamental biological processes at the intersection of vascular biology, immunology, and cellular differentiation.