EGFL7 Antibody, FITC conjugated

What is EGFL7 and why is it a significant target for research?

EGFL7 (Epidermal Growth Factor-like domain 7), also known as VE-statin, is a secreted protein specifically expressed by endothelial cells in normal tissues and by cancer cells in various human tumors . This protein plays critical roles in vascular development and angiogenesis. EGFL7 is particularly significant in oncology research because high levels correlate with higher tumor grade and poorer prognosis . EGFL7 functions as an extracellular matrix-associated protein expressed in activated endothelium, supporting endothelial cell adhesion and protecting endothelial cells from stress-induced apoptosis . Its ability to modulate tumor microenvironment by reducing immune cell infiltration makes it a valuable target for cancer immunotherapy studies . Researchers typically target EGFL7 to understand angiogenic mechanisms and potential therapeutic applications in cancer treatment.

What are the optimal sample preparation protocols for using FITC-conjugated EGFL7 antibody in immunofluorescence?

For effective immunofluorescence with FITC-conjugated EGFL7 antibody, researchers should implement the following protocol:

• Fix tissue sections with paraformaldehyde (PFA) as demonstrated in the literature for EGFL7 staining

• When working with frozen sections, ensure proper cryoprotection to preserve antigen integrity

• During antibody incubation, use a concentration of approximately 0.1 μg of affinity-purified anti-EGFL7 antibody, similar to protocols established for rabbit anti-EGFL7 antibody

• Include appropriate blocking steps (typically 5-10% normal serum from the same species as the secondary antibody) to minimize non-specific binding

• For co-localization studies with endothelial markers like CD31, consider sequential staining approaches to avoid cross-reactivity

• Image using confocal or fluorescence microscopy systems capable of detecting FITC signal (such as Zeiss Z1 Axioimager with Apotome mentioned in the literature)

This approach enables precise localization of EGFL7 in blood vessels, particularly in large vessels with distinct lumen which typically show strong EGFL7 signals .

How can researchers quantitatively analyze EGFL7 expression patterns in tumor vasculature?

Quantitative analysis of EGFL7 expression in tumor vasculature requires a systematic approach:

• Use automated image analysis software (such as Imaris, mentioned in the studies) to quantify microvascular density based on CD31 and EGFL7 co-staining

• Employ a standardized scoring system to categorize blood vessels as EGFL7-positive or negative

• Calculate the percentage of EGFL7-positive intratumoral blood vessels (research shows approximately 25-40% positive vessels in glioma specimens)

• For comparative analysis, use statistical methods to correlate EGFL7 expression with other vascular parameters such as pericyte coverage, vessel maturity, and leakiness

• Consider 3D reconstruction of confocal z-stacks to better visualize the spatial distribution of EGFL7 in complex vascular networks

• For temporal studies, establish consistent imaging parameters across timepoints to enable valid comparisons

This quantitative approach allows researchers to evaluate how EGFL7 expression correlates with vascular morphology, maturity, and functional properties within the tumor microenvironment.

What are the critical considerations when using EGFL7 antibody to study angiogenesis in preclinical tumor models?

When studying angiogenesis with EGFL7 antibody in preclinical models, researchers should address:

• Model selection: Different tumor models exhibit varying degrees of EGFL7 expression and vascular phenotypes. Both cell line-derived xenografts and patient-derived models should be considered

• Timing of analysis: Vascular development is dynamic; therefore, multiple timepoints should be analyzed to capture the evolving role of EGFL7 in vessel formation

• Combined markers: Always co-stain with endothelial markers (CD31) to distinguish EGFL7 expression in tumor vessels versus tumor cells

• Functional correlation: Combine EGFL7 immunostaining with functional vascular assessment using contrast agents (e.g., Gadovist used in studies) to correlate expression with vessel leakiness and maturity

• Treatment effects: When testing anti-angiogenic therapies, compare EGFL7 expression before and after treatment to understand therapy-induced changes

• Validation approaches: Use multiple antibody clones to confirm staining patterns, as demonstrated in studies that achieved 100% overlap with alternative anti-EGFL7 antibodies from different sources

These considerations help ensure robust and reproducible data when investigating EGFL7's role in tumor angiogenesis.

How should researchers design experiments to evaluate the efficacy of anti-EGFL7 antibodies in combination with other anti-angiogenic therapies?

Designing robust experiments to evaluate anti-EGFL7 and anti-angiogenic combination therapies requires:

This comprehensive approach captures the multifaceted effects of targeting EGFL7 in combination with other angiogenesis inhibitors.

What methods are most effective for validating the specificity of EGFL7 antibody staining patterns?

To ensure specificity of EGFL7 antibody staining, researchers should implement:

• Multiple antibody validation: Use alternative anti-EGFL7 antibodies from different sources and confirm signal overlap; 100% overlap between antibodies provides strong validation

• Peptide competition assays: Pre-incubate antibody with excess recombinant EGFL7 protein to demonstrate specific blocking of staining

• Genetic controls: Compare staining in tissues with genetic manipulation of EGFL7 expression (e.g., knockout models or cells with ectopic EGFL7 expression)

• Cross-species reactivity testing: Confirm consistent staining patterns across species if the antibody is designed to recognize conserved epitopes

• Correlation with transcript expression: Validate protein detection with mRNA expression using techniques like qRT-PCR with primers such as 5′-TGCGACGGACACAGAGCCTGCA-3′ and 5′-CAAGTATCTCCCTGCCATCCCA-3′

• Western blot correlation: Confirm antibody specificity by western blot analysis of tissues or cells with varying EGFL7 expression levels

These validation approaches are essential for ensuring the reliability of experimental findings based on EGFL7 immunofluorescence.

How can researchers distinguish between EGFL7 and miR-126 effects when studying vascular phenotypes?

Distinguishing between EGFL7 and miR-126 effects requires specialized approaches:

• Targeted genetic models: Generate models with selective targeting of either EGFL7 coding sequence or miR-126 while preserving the other element

• Rescue experiments: Perform complementation studies where either EGFL7 or miR-126 is reintroduced in knockout models to determine which rescues specific phenotypes

• Expression analysis: Use specific detection methods for each molecule:

  • For miR-126: Employ locked nucleic acid (LNA) probes for in situ hybridization and Taqman MicroRNA Assays utilizing looped RT primers for quantitative analysis

  • For EGFL7 protein: Use validated antibodies that recognize protein-specific epitopes

• Temporal expression mapping: Analyze the developmental timing of expression changes, as EGFL7 and miR-126 may have distinct temporal patterns

• Cell-specific analysis: Determine whether expression patterns differ across cell types, such as between circulating progenitor cells and endothelial cells

• Functional assays: Develop read-outs specific to each molecule's proposed functions to separate their biological effects

This systematic approach helps delineate the contributions of EGFL7 protein versus the intronic miR-126 in complex vascular phenotypes.

What are the technical considerations for using FITC-conjugated EGFL7 antibody in multiparameter flow cytometry?

When incorporating FITC-conjugated EGFL7 antibody in multiparameter flow cytometry, researchers should address:

• Spectral overlap: Account for FITC's emission spectrum (peak ~520nm) when designing panels to minimize spillover with other fluorochromes

• Titration optimization: Determine optimal antibody concentration to achieve maximum signal-to-noise ratio for specific cell populations

• Appropriate controls:

  • Include FMO (fluorescence minus one) controls to establish gating boundaries

  • Use isotype controls conjugated to FITC to assess non-specific binding

• Compensation: Perform proper compensation, especially for adjacent channels (PE, PerCP)

• Cell preparation protocol: For intracellular EGFL7 detection, optimize fixation and permeabilization protocols that preserve both EGFL7 epitopes and surface markers

• Target population identification: Use appropriate markers to identify relevant cell populations, such as:

  • For circulating progenitor cells: CD34ʰⁱCD31ᵈⁱᵐCD45ᵈⁱᵐ cells that express high levels of EGFL7

  • For endothelial cells: CD31ʰⁱCD45⁻ cells

• Data analysis approach: Implement consistent gating strategies across experiments and consider dimensional reduction techniques for complex datasets

These considerations ensure reliable detection of EGFL7-expressing cells in heterogeneous populations.

How can EGFL7 antibodies be used to identify and quantify circulating progenitor cells as biomarkers for anti-angiogenic therapy?

EGFL7 antibodies can be effectively implemented for CPC biomarker analysis through:

This approach leverages CPCs as a pharmacodynamic marker for interrogating in vivo activities of anti-EGFL7 therapeutics, as demonstrated in both preclinical models and phase I clinical trials .

What experimental approaches best characterize the role of EGFL7 in stress-induced endothelial cell responses?

To effectively characterize EGFL7's role in endothelial stress responses, researchers should employ:

• In vitro stress models: Subject endothelial cells to relevant stressors:

  • Serum starvation to mimic nutrient deprivation

  • Hypoxic conditions to represent tumor microenvironment

  • Inflammatory cytokine exposure

• Survival assays: Quantify stress-induced apoptosis with and without:

  • Recombinant EGFL7 protein supplementation

  • Anti-EGFL7 antibody treatment

  • Combination with anti-VEGF agents

• Adhesion analysis: Assess endothelial cell adhesion to extracellular matrix components in the presence of:

  • Purified EGFL7

  • Fibronectin

  • EGFL7/fibronectin combinations

  • Specific blocking antibodies against integrin α5β1

• Structural studies: Employ transmission electron microscopy to analyze EGFL7 oligomerization and fibrillar structures, which provide insight into functional mechanisms

• Sprouting assays: Utilize 3D cell culture models to evaluate capillary-like structure formation under various conditions

• Molecular pathway analysis: Investigate signaling cascades activated by EGFL7 during stress response using phospho-protein analysis

These approaches provide comprehensive characterization of how EGFL7 functions to protect endothelial cells under stress conditions relevant to tumor vasculature.