atf31 Antibody

What is CD31/PECAM-1 and what cellular structures does it target?

CD31 (Platelet Endothelial Cell Adhesion Molecule-1 or PECAM-1) is a transmembrane glycoprotein expressed primarily on endothelial cells, platelets, and various leukocytes. It functions as an adhesion molecule and signaling receptor involved in vascular biology. CD31/PECAM-1 antibodies specifically target the cell membrane of endothelial cells, making them excellent markers for blood vessels and vascular structures. In research applications, CD31/PECAM-1 antibody has been demonstrated to localize specifically to the cell membrane of endothelial cells, as evidenced by immunofluorescence studies of the bEnd.3 mouse endothelioma cell line . The antibody recognizes the extracellular domain (Glu18-Lys590) of CD31/PECAM-1, allowing for detection of this protein in its native conformation on the cell surface .

What species reactivity does CD31/PECAM-1 antibody demonstrate?

CD31/PECAM-1 antibody demonstrates cross-reactivity with human, mouse, and rat samples, making it versatile for comparative studies across species. This cross-reactivity has been validated through multiple experimental techniques. Flow cytometry has confirmed reactivity with mouse and rat splenocytes . Western blot analyses have demonstrated detection of human CD31/PECAM-1 in human microvascular endothelial cells (HMVECs) . Immunofluorescence microscopy has confirmed detection of CD31/PECAM-1 in mouse heart tissues . This multi-species reactivity is particularly valuable for translational research where findings in rodent models can be correlated with human samples.

What are the optimal sample preparation techniques for CD31/PECAM-1 detection?

Optimal sample preparation for CD31/PECAM-1 detection varies by application technique:

For flow cytometry:

• For whole blood samples: Use targeted lysis of red blood cells while preserving leukocyte populations

• For isolated cell populations: Maintain cells in PBS with 0.5-1% BSA

• Optimal staining occurs with fresh samples processed at 4°C to prevent receptor internalization

For immunofluorescence/immunohistochemistry:

• Fixation with 4% paraformaldehyde preserves epitope accessibility

• Tissue sections benefit from antigen retrieval techniques

• Optimal concentration has been determined as 10 μg/mL when applied for 3 hours at room temperature

• Counterstaining with nuclear dyes such as DAPI enhances visualization of cellular architecture

For western blotting:

• Standard protein extraction protocols are effective

• Both reducing and non-reducing conditions preserve epitope recognition

• Samples should be fresh or properly stored at -80°C to prevent protein degradation

What controls should be included in CD31/PECAM-1 antibody experiments?

Rigorous experimental design requires appropriate controls for CD31/PECAM-1 antibody applications:

For flow cytometry:

• Isotype control antibody (such as AB-108-C) is essential to determine non-specific binding and establish proper gating

• Unstained samples help establish autofluorescence baseline

• Known positive controls (endothelial cells) and negative controls (epithelial cell lines) validate staining specificity

For immunofluorescence:

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

• Isotype controls at matching concentrations evaluate primary antibody specificity

• Competitive blocking with recombinant CD31/PECAM-1 confirms epitope specificity

For western blotting:

• Loading controls (β-actin) are essential for normalization of protein quantity

• Molecular weight markers confirm expected protein size

• Positive control samples (endothelial cell lysates) validate antibody functionality

How can CD31/PECAM-1 antibody be used to investigate endothelial-to-mesenchymal transition (EndMT)?

CD31/PECAM-1 antibody serves as a critical tool for investigating endothelial-to-mesenchymal transition (EndMT), a process implicated in fibrotic diseases and cardiac dysfunction. In EndMT research, CD31/PECAM-1 functions as an endothelial marker that diminishes as cells transition to a mesenchymal phenotype.

Methodological approach for EndMT investigation:

• Establish baseline CD31/PECAM-1 expression in control endothelial cells using western blot or immunofluorescence

• Induce EndMT through TGF-β2 treatment or other stimuli

• Monitor the concurrent decrease in CD31/PECAM-1 expression and increase in mesenchymal markers (SM22α, FSP1, α-SMA)

• Quantify changes through densitometric analysis of western blots

• Confirm cellular phenotypic changes through immunofluorescence co-staining

Research has demonstrated that during EndMT, CD31 expression decreases while mesenchymal markers increase. This transition can be modulated by factors such as AcSDKP and FGF2, which preserve CD31 expression even in the presence of TGF-β2 . For example, human microvascular endothelial cells (HMVECs) treated with TGF-β2 showed reduced CD31 expression, but preincubation with AcSDKP prevented this reduction .

What is the relationship between CD31/PECAM-1 and the TGF-β/Smad signaling pathway?

CD31/PECAM-1 expression is inversely related to TGF-β/Smad signaling activation, a key pathway in fibrotic processes and tissue remodeling. Research using CD31/PECAM-1 antibody has elucidated this regulatory relationship:

• TGF-β2 treatment of endothelial cells activates Smad3 phosphorylation (p-Smad3) while decreasing CD31/PECAM-1 expression

• CD31/PECAM-1 and p-Smad3 double-labeled immunofluorescence reveals cells undergoing active TGF-β signaling lose endothelial characteristics

• Interventions that inhibit p-Smad3 (such as AcSDKP) maintain CD31/PECAM-1 expression levels

How does CD31/PECAM-1 expression correlate with neovascularization after stroke?

CD31/PECAM-1 antibody provides valuable insights into post-stroke neovascularization processes. Research using this antibody has revealed several key findings:

• CD31 immunofluorescence allows quantification of microvascular density in the peri-infarct cortex

• Increased CD31-positive microvessels correlate with functional recovery after stroke

• Neutrophil depletion (using anti-Ly6G antibody) increases CD31-positive microvascular density in the peri-infarct cortex compared to control antibody treatment

Quantitative analysis of microvascular density using CD31 staining has shown that interventions affecting neutrophil activity significantly impact neovascularization. For example, studies demonstrated that mice treated with anti-Ly6G antibody showed increased microvascular density compared to those treated with control antibody . This finding was further validated by in-vivo multiphoton microscopy showing increased perfused capillary length in the treated group, indicating functional neovascularization .

The correlation between CD31-positive vessel density and functional outcomes provides a valuable biomarker for therapeutic efficacy in stroke recovery research.

Can CD31/PECAM-1 antibody effectively assess blood-brain barrier (BBB) integrity?

CD31/PECAM-1 antibody serves as an effective tool for assessing blood-brain barrier (BBB) integrity when used in conjunction with markers of vascular leakage. Research methodology for BBB assessment includes:

• CD31 immunofluorescence to identify vascular structures

• Co-staining with IgG to detect extravascular deposits indicating BBB breakdown

• Quantification of IgG extravascular deposits relative to CD31-positive vessels

• Correlation of BBB disruption with pathological processes or therapeutic interventions

Studies have demonstrated that neutrophil depletion reduces BBB breakdown after stroke, as evidenced by reduced IgG extravascular deposits in the peri-infarct cortex . Quantitative analysis revealed significantly lower IgG extravascular deposits in anti-Ly6G antibody-treated mice compared to control antibody-treated mice . This methodology provides a powerful approach for assessing therapeutic strategies aimed at preserving BBB integrity in neurological disorders.

What are the optimal dilutions and conditions for CD31/PECAM-1 antibody across different applications?

Optimal conditions for CD31/PECAM-1 antibody application vary by technique:

Flow Cytometry:

• Concentration: Titration typically optimal between 0.25-1.0 μg per 10^6 cells

• Buffer: PBS with 0.5-1% BSA and 0.1% sodium azide

• Incubation: 30 minutes at 4°C

• Secondary detection: Fluorophore-conjugated anti-goat IgG (e.g., Allophycocyanin-conjugated or Phycoerythrin-conjugated)

Immunofluorescence/Immunohistochemistry:

• Concentration: 10 μg/mL demonstrated optimal signal-to-noise ratio

• Incubation: 3 hours at room temperature or overnight at 4°C

• Detection: Fluorophore-conjugated secondary antibodies (e.g., NorthernLights™ 493-conjugated Anti-Goat IgG)

• Counterstain: DAPI for nuclear visualization

Western Blotting:

• Concentration: Requires empirical determination based on sample type and detection method

• Blocking: Standard blocking buffers (5% non-fat dry milk or BSA)

• Normalization: β-actin serves as an appropriate loading control

For each application, researchers should perform preliminary titration experiments to determine optimal antibody concentration for their specific experimental system.

How should CD31/PECAM-1 antibody be validated for experimental use?

Comprehensive validation of CD31/PECAM-1 antibody should include:

• Specificity testing:

  • Comparison with isotype control antibody (e.g., AB-108-C)

  • Competitive blocking with recombinant CD31/PECAM-1 protein

  • Absence of staining in known negative cell types

• Sensitivity assessment:

  • Titration experiments to determine minimal effective concentration

  • Signal-to-noise ratio optimization

  • Detection limit determination using serial dilutions of positive controls

• Cross-reactivity verification:

  • Testing across multiple species (human, mouse, rat) if cross-reactivity is claimed

  • Comparison with species-specific antibodies

  • Sequence alignment analysis of epitope regions

• Application-specific validation:

  • For flow cytometry: Comparison with established CD31 antibody clones

  • For western blotting: Confirmation of expected molecular weight

  • For immunohistochemistry: Correlation with known vascular patterns

Validation should be documented with appropriate positive and negative controls to ensure reproducible experimental outcomes.

What quantification methods are recommended for CD31/PECAM-1 expression analysis?

Quantification methods for CD31/PECAM-1 expression depend on the experimental technique:

Flow Cytometry Quantification:

• Percent positive cells relative to isotype control

• Mean/median fluorescence intensity (MFI) for expression level assessment

• Standardization using calibration beads for inter-experimental comparison

Immunofluorescence Quantification:

• Microvascular density (MVD) measurement by counting CD31-positive vessels per field

• Assessment of vessel morphology (diameter, branching)

• Colocalization analysis with other markers using Pearson's or Manders' coefficients

• For each tissue section, images from multiple fields of view (at least six) at 400× magnification should be evaluated

Western Blot Quantification:

• Densitometric analysis normalized to loading controls (β-actin)

• Relative quantification compared to control samples

• Multiple independent experiments (n≥3) for statistical validity

For diabetic heart studies, quantification has included counting CD31 and FGFR1 double-labeled cells or CD31 and p-MAP4K4 double-labeled cells per visual field using fluorescence microscopy . Similarly, CD31 and α-SMA double-labeled cells can be quantified to assess EndMT in tissue samples.

How can non-specific binding of CD31/PECAM-1 antibody be reduced?

Non-specific binding of CD31/PECAM-1 antibody can compromise experimental results. Implement these strategies to minimize background:

• Optimize blocking protocols:

  • Extend blocking time to 1-2 hours at room temperature

  • Use species-appropriate serum (5-10%) matched to secondary antibody

  • Add 0.1-0.3% Triton X-100 for intracellular staining to reduce hydrophobic interactions

• Improve washing procedures:

  • Increase wash buffer volume and duration

  • Use gentle agitation during washing steps

  • Add 0.05-0.1% Tween-20 to wash buffers to reduce non-specific interactions

• Optimize antibody concentration:

  • Perform titration experiments to determine minimal effective concentration

  • Compare signal-to-noise ratio across concentration range

  • Include isotype controls at matching concentrations

• Modify sample preparation:

  • Ensure complete fixation without over-fixation

  • Consider alternative fixatives if formalin-fixed samples show high background

  • Implement antigen retrieval optimization for tissue sections

Including appropriate isotype control antibodies (such as AB-108-C) in parallel with CD31/PECAM-1 antibody allows accurate assessment of specific versus non-specific binding .

What steps should be taken when CD31/PECAM-1 antibody shows weak or no signal?

When CD31/PECAM-1 antibody yields weak or absent signal, systematic troubleshooting should include:

• Antibody functionality verification:

  • Test antibody on known positive control (e.g., endothelial cell line)

  • Verify antibody hasn't exceeded recommended storage time

  • Check for signs of antibody denaturation (precipitation, cloudiness)

• Epitope accessibility improvement:

  • Implement or optimize antigen retrieval methods

  • Consider alternative fixation protocols

  • Increase permeabilization for intracellular domains

• Detection system enhancement:

  • Utilize signal amplification methods (tyramide signal amplification, polymer-based detection)

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

  • Optimize secondary antibody concentration and incubation conditions

• Antibody concentration adjustment:

  • Increase antibody concentration incrementally

  • Compare results across concentration range (5-20 μg/mL for immunofluorescence)

• Sample quality assessment:

  • Verify target protein expression in sample type

  • Check for protein degradation in stored samples

  • Assess tissue morphology and preservation

For immunofluorescence applications, optimization might require adjusting the antibody concentration from the standard 10 μg/mL depending on specific tissue characteristics and fixation conditions .

How can contradictory CD31/PECAM-1 expression data be analyzed and reconciled?

Contradictory CD31/PECAM-1 expression data requires systematic analysis and reconciliation:

• Methodological comparison:

  • Evaluate different detection techniques (western blot vs. immunofluorescence vs. flow cytometry)

  • Compare sensitivity and specificity of each method

  • Assess whether contradictions stem from qualitative versus quantitative differences

• Biological context analysis:

  • Consider developmental stage or disease progression impact

  • Evaluate microenvironmental factors affecting expression

  • Assess potential post-translational modifications affecting epitope recognition

• Experimental variables assessment:

  • Compare fixation and processing methods

  • Evaluate antibody clones and epitopes recognized

  • Consider species differences in CD31/PECAM-1 expression patterns

• Data integration approach:

  • Implement multiple complementary techniques

  • Use quantitative methods with appropriate statistical analysis

  • Consider single-cell approaches to resolve population heterogeneity

Research on diabetic hearts demonstrated that CD31 expression patterns must be interpreted in context of other markers. For example, CD31/α-SMA double-positive cells indicate EndMT processes, while CD31/p-Smad3 coexpression indicates active TGF-β signaling in endothelial cells . These contextual analyses help reconcile seemingly contradictory findings about CD31 expression in complex disease models.

How should CD31/PECAM-1 quantification be standardized across different experiments?

Standardization of CD31/PECAM-1 quantification across experiments requires systematic approaches:

For immunofluorescence quantification, researchers should standardize by examining a minimum of six different fields of view at 400× magnification for each tissue section as demonstrated in studies of CD31 expression in diabetic hearts . For western blot analysis, densitometric quantification should be normalized to appropriate loading controls such as β-actin, with representative blots from multiple independent experiments (typically n=3 or n=4) .

What insights can CD31/PECAM-1 provide about endothelial dysfunction in disease models?

CD31/PECAM-1 antibody staining reveals critical insights about endothelial dysfunction across disease models:

In diabetic cardiomyopathy:

• CD31/PECAM-1 staining demonstrates reduced microvascular density

• Co-staining with mesenchymal markers (α-SMA, SM22α) reveals EndMT

• Double-labeling with CD31 and p-Smad3 indicates increased TGF-β signaling in endothelial cells

In stroke models:

• CD31/PECAM-1 staining quantifies neovascularization in peri-infarct regions

• Combined with IgG staining, CD31 helps assess BBB integrity

• CD31-positive vessel density correlates with functional recovery

In vascular remodeling:

• CD31/PECAM-1 antibody visualizes altered microvascular architecture

• Quantification of CD31-positive vessels provides measure of angiogenic responses

• Perfusion studies with dextran combined with CD31 staining differentiates between functional and non-functional vessels

Research has demonstrated that AcSDKP treatment in diabetic mice restored endothelial FGFR1 and P-MAP4K4 expression, reduced EndMT (indicated by decreased CD31/α-SMA double-positive cells), and decreased TGF-β signaling (shown by reduced CD31/p-Smad3 double-positive cells) .

How can CD31/PECAM-1 antibody be integrated into multiplex staining protocols?

Integration of CD31/PECAM-1 antibody into multiplex staining requires careful protocol optimization:

Multiplex Panel Design:

Multiplex Staining Procedure:

• Optimize fixation protocol compatible with all antibodies

• Implement sequential staining if antibody species overlap

• Include appropriate blocking steps between antibody applications

• Carefully select non-overlapping fluorophores

• Include single-stain controls for spectral compensation

Research has successfully employed multiple multiplex combinations:

• CD31/FGFR1 and CD31/P-MAP4K4 double immunofluorescence in diabetic heart tissue

• CD31/α-SMA, VE-cadherin/SM22α, and CD31/p-Smad3, to assess EndMT processes

• CD31 combined with Ly6G to visualize neutrophil-vessel interactions in stroke models

These multiplex approaches provide contextual information about CD31-positive cells that would not be evident from single-marker staining.

How is CD31/PECAM-1 antibody utilized in neurovascular research?

CD31/PECAM-1 antibody serves as a fundamental tool in neurovascular research, particularly in studying stroke pathophysiology and recovery:

• Blood-Brain Barrier (BBB) integrity assessment:

  • CD31 staining identifies vascular structures

  • Co-staining with extravasated IgG quantifies BBB breakdown

  • Studies demonstrate that neutrophil depletion reduces BBB breakdown as measured by decreased IgG extravascular deposits in the peri-infarct cortex

• Neovascularization quantification:

  • CD31-positive microvessels are counted in peri-infarct regions

  • Microvascular density measurement provides quantitative assessment of angiogenic response

  • Research shows increased CD31-positive vessel density following anti-Ly6G antibody treatment compared to control antibody treatment

• Functional vessel assessment:

  • CD31 immunostaining combined with in-vivo multiphoton microscopy of FITC-dextran perfused vessels

  • This combined approach distinguishes between structural and functional vessels

  • Quantification of perfused capillary length provides functional assessment of neovascularization

• Neutrophil-vessel interaction analysis:

  • Co-staining of Ly6G-labeled neutrophils with CD31-positive microvessels

  • Visualization of neutrophils within brain vessels and in parenchyma

  • Research has demonstrated neutrophil accumulation in the brain during all stages of ischemic stroke

These applications have advanced understanding of neurovascular remodeling after stroke and identified potential therapeutic targets for enhancing recovery.

What role does CD31/PECAM-1 antibody play in studying diabetic cardiovascular complications?

CD31/PECAM-1 antibody is instrumental in investigating diabetic cardiovascular complications through several applications:

• Endothelial-to-mesenchymal transition (EndMT) assessment:

  • CD31/α-SMA double immunofluorescence identifies endothelial cells transitioning to mesenchymal phenotype

  • Quantification of CD31/α-SMA double-positive cells measures EndMT in diabetic hearts

  • Research demonstrates increased EndMT in diabetic hearts compared to controls

• Signaling pathway investigation:

  • CD31/p-Smad3 co-staining reveals TGF-β signaling activation in endothelial cells

  • CD31/FGFR1 and CD31/P-MAP4K4 double immunofluorescence assesses protective signaling pathways

  • Studies show AcSDKP treatment restores FGFR1 and P-MAP4K4 levels in diabetic hearts

• Therapeutic intervention evaluation:

  • CD31 staining quantifies vascular preservation following treatment

  • Comparison of microvascular density between treatment groups

  • Research demonstrates AcSDKP inhibits TGF-β/Smad signaling and EndMT in diabetic hearts

• Correlation with molecular markers:

  • Western blot analysis of CD31 levels correlates with immunofluorescence findings

  • Integration of protein expression data with histological observations

  • Studies show concordance between reduced CD31 expression and increased mesenchymal markers in diabetic hearts