CD31 FITC Antibody

What is CD31 and why is it an important research target?

CD31, also known as platelet-endothelial cell adhesion molecule-1 (PECAM-1), is a 130-140 kDa transmembrane glycoprotein belonging to the immunoglobulin superfamily. Its significance in research stems from its widespread expression on vascular endothelial cells and, at lower levels, on leukocytes and platelets. CD31 plays critical roles in cell adhesion, transendothelial migration, and signal transduction, making it an important marker for studying vascular biology, inflammation, and immune responses. The protein's involvement in wound healing, angiogenesis, and cellular migration in inflammatory conditions further highlights its research importance.

What cell types express CD31 and at what relative levels?

CD31 is most abundantly expressed on endothelial cells, where it comprises a significant portion of intercellular junctions. It is also present, albeit at lower expression levels, on various hematopoietic and immune cells including platelets, monocytes, neutrophils, natural killer cells, megakaryocytes, and specific T-cell subsets. This differential expression pattern makes CD31 particularly valuable for identifying and isolating endothelial cells, as well as studying endothelial-immune cell interactions in various physiological and pathological contexts.

How does FITC conjugation affect CD31 antibody functionality?

FITC conjugation provides a fluorescent tag that enables detection of CD31 without significantly altering the antibody's binding specificity or affinity when properly executed. The FITC fluorophore is excited by blue laser (488 nm) and emits at approximately 520-530 nm, making it compatible with standard flow cytometry and fluorescence microscopy equipment. Researchers should note that FITC conjugation may marginally reduce binding affinity compared to unconjugated antibodies in some applications, but the validated clones described in the literature have been optimized to maintain functionality while providing reliable fluorescent detection.

What are the primary research applications for CD31 FITC antibodies?

CD31 FITC antibodies are primarily employed in flow cytometry for identifying and quantifying endothelial cells and specific leukocyte populations. They are also widely used in immunohistochemical staining of frozen tissue sections and immunofluorescence microscopy of cultured cells. These applications enable researchers to study vascular development, angiogenesis, inflammation, immune cell trafficking, and endothelial cell junction integrity. The antibodies can be used in both in vitro systems and ex vivo analysis of clinical samples, providing versatility across multiple research contexts.

What are the optimal titration strategies for CD31 FITC antibodies in flow cytometry?

Optimal titration of CD31 FITC antibodies is essential for achieving high signal-to-noise ratios while conserving reagents. Begin with manufacturer-recommended concentrations (typically ≤1 μg per test or 5 μL per 10^6 cells in 100 μL suspension) and perform a titration series using 2-fold dilutions above and below this concentration. Evaluate signal intensity, separation between positive and negative populations, and background staining to determine optimal concentration. Cell numbers should be empirically determined but typically range from 10^5 to 10^8 cells per test. Remember that optimal concentrations may vary between different biological samples (e.g., cell lines versus primary cells) and different experimental conditions, necessitating validation for each specific application.

How should researchers optimize CD31 FITC antibody staining for immunohistochemistry?

For immunohistochemical applications with frozen tissue sections, researchers should start with antibody concentrations of ≤20 μg/mL and optimize through serial dilutions. Critical considerations include: (1) Proper tissue fixation—typically 4% paraformaldehyde for 10-15 minutes preserves CD31 epitopes while maintaining tissue architecture; (2) Adequate blocking with 5-10% serum from the same species as the secondary antibody to reduce non-specific binding; (3) Optimal incubation conditions—typically 1-2 hours at room temperature or overnight at 4°C; (4) Inclusion of appropriate positive and negative controls to validate staining specificity; and (5) Protection from light during and after staining to prevent photobleaching of the FITC fluorophore. Counterstaining with DAPI for nuclear visualization can enhance interpretation of vascular structures.

What are the key considerations for multi-color flow cytometry panels including CD31 FITC?

When designing multi-color panels incorporating CD31 FITC antibodies, researchers should consider: (1) Spectral overlap—FITC emission spectrum overlaps with PE and other fluorophores, requiring proper compensation; (2) Expression levels—CD31's high expression on endothelial cells may require adjustment of voltages or antibody concentration compared to markers with lower expression; (3) Marker co-expression patterns—CD31 co-staining with markers like CD34, VEGFR2, or CD45 can help discriminate between endothelial cells and hematopoietic cells; (4) Viability dyes—inclusion of viability markers helps exclude dead cells that may bind antibodies non-specifically; and (5) Isotype controls—inclusion of appropriate isotype-matched control antibodies conjugated to FITC to establish specificity of staining. Fluorophore brightness hierarchies should align with target abundance (brighter fluorophores for less abundant targets).

How can researchers optimize storage conditions to maintain CD31 FITC antibody performance?

To maintain optimal performance of CD31 FITC antibodies, store at 2-8°C and protect from prolonged light exposure, which can cause photobleaching and significantly reduce signal intensity. Never freeze FITC-conjugated antibodies as freeze-thaw cycles can cause protein denaturation and fluorophore degradation. Aliquoting stock solutions into single-use volumes minimizes repeated freezing and thawing. For long-term storage, add protein stabilizers such as 1% BSA if not already present in the storage buffer. Monitor antibody performance periodically by testing with positive control samples, and note that most FITC-conjugated antibodies maintain stability for approximately 12 months when stored under recommended conditions. Upon signs of reduced performance, such as diminished staining intensity or increased background, replace with fresh antibody preparations.

How can CD31 FITC antibodies be effectively utilized to study transendothelial migration?

For studying transendothelial migration (TEM), researchers can implement advanced experimental designs using CD31 FITC antibodies. Methodologically, establish endothelial monolayers on Transwell inserts and measure leukocyte trafficking across this barrier under various conditions. CD31 FITC staining allows visualization of the dynamic redistribution of CD31 during this process, particularly to the lateral border recycling compartment (LBRC). Time-lapse confocal microscopy with CD31 FITC can track the accumulation of CD31 around transmigrating leukocytes. For quantitative analysis, researchers should evaluate JAM-A/CD31 co-localization using dual immunofluorescence and calculate Pearson's correlation coefficients. Specific blocking antibodies against different CD31 domains can determine which homophilic interactions are critical for the diapedesis process. This approach reveals how tyrosine-660 phosphorylation status affects CD31 trafficking and leukocyte migration, providing insights into inflammatory responses and potential therapeutic targets.

What are the methodological approaches for studying CD31-mediated angiogenesis using FITC-conjugated antibodies?

To investigate CD31's role in angiogenesis, researchers can employ several methodological strategies using CD31 FITC antibodies. In vitro tube formation assays on Matrigel with endothelial cells can be monitored via time-lapse fluorescence microscopy after CD31 FITC staining, quantifying parameters such as tube length, branch points, and network complexity. For ex vivo approaches, aortic ring assays combined with CD31 FITC immunofluorescence enable visualization of sprouting vessels. In vivo, researchers can utilize Matrigel plug assays or tumor xenograft models followed by tissue harvesting and CD31 FITC staining to quantify vessel density, branching, and diameter. Advanced analytical techniques include 3D reconstruction of confocal z-stacks to visualize vascular networks comprehensively. Flow cytometric analysis of CD31 FITC-labeled cells isolated from these models can quantify endothelial cell proliferation and apoptosis rates when combined with appropriate cell cycle or apoptosis markers.

How can researchers methodologically investigate the signaling functions of CD31 using FITC-labeled antibodies?

Investigating CD31 signaling functions requires sophisticated approaches beyond simple detection. Researchers can use CD31 FITC antibodies to perform co-immunoprecipitation experiments followed by western blotting to identify signaling partners. Proximity ligation assays (PLA) combined with CD31 FITC staining can visualize protein-protein interactions in situ. For studying dynamic signaling, fluorescence resonance energy transfer (FRET) between CD31 FITC and acceptor fluorophore-labeled signaling molecules can detect conformational changes and interactions in real-time. Quantitative phosphoproteomic analysis following CD31 cross-linking can identify downstream signaling cascades, particularly focusing on ERK1/2 activation pathways. Researchers should implement proper controls including non-stimulating antibodies and phosphatase inhibitors when studying phosphorylation-dependent events. Flow cytometric analysis of intracellular signaling molecules (phospho-flow) following CD31 engagement provides single-cell resolution of signaling events in heterogeneous populations.

How should researchers analyze and interpret heterogeneous CD31 expression patterns in flow cytometry?

When analyzing heterogeneous CD31 expression patterns, researchers should implement a multi-parameter analytical approach. First, establish clear gating strategies based on forward/side scatter properties to define cell populations of interest before examining CD31 FITC intensity. Utilize bivariate plots of CD31 versus lineage-specific markers (CD45, CD34, etc.) to distinguish between endothelial cells (CD31^high^CD45^-^), platelets (CD31^+^CD41^+^), and leukocyte subsets (CD31^low^CD45^+^). Histogram overlays comparing sample staining with isotype controls should be generated to visualize the distribution of expression intensities. Quantify results using appropriate statistical measures including median fluorescence intensity (MFI) rather than mean values when distributions are non-Gaussian. For more complex patterns, consider computational approaches such as viSNE or FlowSOM to visualize high-dimensional data. When comparing samples, use standardized beads to ensure consistent instrument settings across experiments, enabling reliable inter-experimental comparisons.

What are the methodological approaches to resolving contradictory findings when using CD31 FITC antibodies?

When faced with contradictory results using CD31 FITC antibodies, implement a systematic troubleshooting methodology. First, validate antibody specificity by comparing multiple antibody clones (e.g., 390, TLD-3A12, 158-2B3) targeting different CD31 epitopes. Perform blocking experiments with unconjugated antibodies to confirm specific binding. Control for technical variability by standardizing sample preparation, fixation protocols, and instrument settings. Consider epitope masking due to protein-protein interactions or conformation changes that might affect antibody access in different experimental contexts. Investigate potential splice variants or post-translational modifications of CD31 that might be differentially recognized by various antibodies. Perform correlation analyses between flow cytometry data and other detection methods such as qPCR or western blotting to validate findings. Finally, consider biological variables such as cell activation state, culture conditions, or tissue microenvironment that may genuinely cause differential CD31 expression or localization.

How can researchers quantitatively assess CD31 expression in complex tissue microenvironments?

For quantitative assessment of CD31 in complex tissues, implement advanced image analysis methodologies. Begin with high-resolution confocal microscopy of tissue sections stained with CD31 FITC antibodies and appropriate counterstains. Utilize automated image analysis software (ImageJ/FIJI with appropriate plugins, CellProfiler, or commercial platforms) to segment CD31-positive structures and quantify parameters including vessel density, diameter, branching patterns, and total vascular area. For more sophisticated analysis, employ machine learning algorithms to classify different vascular structures based on morphological features. Use multi-spectral imaging to distinguish CD31 FITC signal from tissue autofluorescence, particularly in lipid-rich or fibrous tissues. For comparative studies, normalize CD31 expression to tissue area or volume, and consider using tissue microarrays to standardize staining conditions across multiple samples. Vessel co-option in tumors can be distinguished from neoangiogenesis by combining CD31 staining with basement membrane markers like collagen IV.

What methodological approaches can address weak or variable CD31 FITC staining in flow cytometry?

When encountering weak or inconsistent CD31 FITC staining in flow cytometry, implement a systematic optimization strategy. Begin by testing fresh antibody preparations as FITC is susceptible to photobleaching and degradation over time. Optimize cell preparation protocols by ensuring gentle dissociation methods that preserve surface epitopes—enzymatic digestions should be carefully titrated as excessive protease exposure can cleave CD31. Extend antibody incubation times (30-60 minutes at 4°C) and consider using staining buffers containing sodium azide to prevent receptor internalization during processing. For tissues with inherently low CD31 expression, signal amplification systems such as biotinylated anti-FITC antibodies followed by streptavidin-conjugated fluorophores can enhance detection sensitivity. Evaluate different permeabilization protocols if studying intracellular pools of CD31. Finally, consider alternative, brighter fluorophores like PE or APC if FITC signal remains suboptimal after these optimizations.

How can researchers methodologically distinguish between specific and non-specific binding of CD31 FITC antibodies?

To differentiate between specific and non-specific binding, implement a comprehensive validation approach. Include multiple control samples: (1) unstained cells to establish autofluorescence baseline; (2) isotype-matched FITC-conjugated control antibodies of the same host species and immunoglobulin class to identify Fc receptor-mediated binding; (3) blocking experiments with excess unconjugated CD31 antibody before adding CD31 FITC to confirm epitope-specific binding; and (4) CD31-negative cell lines as biological negative controls. Titrate antibodies carefully to determine the concentration that maximizes the positive-to-negative signal ratio rather than absolute signal intensity. Implement doublet discrimination in flow cytometry analysis to exclude cell aggregates that can give false positive signals. For frozen tissue sections, include absorption controls where the antibody is pre-incubated with recombinant CD31 protein before staining to block specific binding sites. Finally, compare staining patterns across multiple CD31 antibody clones targeting different epitopes to confirm consistent localization patterns.

What are the optimal fixation and permeabilization protocols for CD31 FITC immunofluorescence studies?

Optimizing fixation and permeabilization is critical for CD31 FITC immunofluorescence applications. For cell surface CD31 detection, implement mild fixation with 2-4% paraformaldehyde for 10-15 minutes at room temperature, which preserves epitope structure while maintaining membrane integrity. Avoid methanol fixation, which can denature the CD31 protein and destroy epitope recognition. For intracellular or junction-associated CD31 pools, after paraformaldehyde fixation, apply gentle permeabilization using 0.1-0.3% Triton X-100 or 0.1% saponin for 5-10 minutes. Saponin is particularly suitable for visualizing membrane-associated proteins as it creates smaller pores than Triton. For delicate samples like primary endothelial cells, consider using digitonin (10-50 μg/mL) for selective plasma membrane permeabilization while preserving intracellular membranes. After fixation and permeabilization, implement a thorough blocking step (5-10% normal serum with 1% BSA) for at least 30 minutes to minimize background staining. Always process experimental and control samples under identical conditions to ensure comparability.