FLT4 Antibody, HRP conjugated

What is FLT4/VEGFR3 and what are its primary biological functions?

FLT4, also known as VEGFR3 (Vascular Endothelial Growth Factor Receptor 3), is a tyrosine kinase receptor that acts as a cell-surface receptor for VEGFC and VEGFD growth factors. This receptor plays essential roles in adult lymphangiogenesis and in the development of the vascular network and cardiovascular system during embryonic development . FLT4 mediates critical cellular functions including:

• Promotion of proliferation, survival, and migration of endothelial cells

• Regulation of angiogenic sprouting

• Enhancement of VEGFC production through positive feedback loops

• Modulation of KDR signaling through heterodimer formation

• Activation of multiple signaling cascades including MAPK1/ERK2, MAPK3/ERK1, MAPK8/JUN, and AKT1 pathways

The protein is particularly important for lymphatic endothelium maintenance, and mutations in the FLT4 gene can cause hereditary lymphedema type IA .

How does HRP conjugation affect antibody applications for FLT4 detection?

HRP (Horseradish Peroxidase) conjugation to FLT4 antibodies provides significant advantages for various detection methods:

• Direct detection capability: Eliminates the need for secondary antibodies, reducing background and non-specific binding

• Enhanced sensitivity: The enzymatic amplification provided by HRP improves detection of low-abundance FLT4 in samples

• Compatibility with multiple substrates: Works with colorimetric (DAB, TMB), chemiluminescent, and fluorescent substrates

• Application versatility: Particularly effective for Western blotting (recommended dilution 1:500) and immunohistochemistry

What experimental applications are most suitable for FLT4 antibodies with HRP conjugation?

FLT4 antibodies with HRP conjugation are particularly effective for several research applications:

When working with HRP-conjugated FLT4 antibodies, researchers should be aware that the detection system must be compatible with HRP enzymatic activity and that proper controls should be included to account for potential endogenous peroxidase activity in tissue samples.

What are the optimal storage and handling conditions for preserving FLT4 antibody activity?

To maintain optimal activity of HRP-conjugated FLT4 antibodies:

• Storage temperature: Store at -20°C as received to maintain activity

• Buffer composition: Typically provided in PBS (pH 7.3) containing 1% BSA and 50% glycerol

• Stability: Remains stable for approximately 12 months from date of receipt when properly stored

• Shipping conditions: Usually shipped on blue ice to maintain activity

• Aliquoting: Divide into single-use aliquots to avoid repeated freeze-thaw cycles

• Light exposure: Minimize exposure to light, particularly important for fluorescent applications

• Working dilutions: Prepare fresh working dilutions on the day of use

• Contamination prevention: Use sterile techniques when handling to prevent microbial contamination

Adhering to these guidelines helps maintain antibody reactivity and ensures reproducible experimental results.

How can researchers optimize detection of FLT4/VEGFR3 in lymphatic endothelial cells from heterogeneous tissue samples?

Optimizing FLT4/VEGFR3 detection in complex tissue samples requires careful consideration of multiple factors:

Tissue preparation protocol:

• For FFPE samples: Use heat-induced epitope retrieval (citrate buffer pH 6.0 or Tris-EDTA pH 9.0)

• For frozen sections: Brief fixation with 4% paraformaldehyde preserves epitope accessibility

Multi-marker approach for lymphatic vessel identification:
Researchers should employ a panel of lymphatic markers alongside FLT4/VEGFR3 for accurate identification:

Signal amplification strategies:

• Tyramide signal amplification can enhance detection sensitivity

• Optimization of antibody concentration (starting with 1:500 dilution)

• Use of appropriate blocking (5-10% serum from the same species as secondary antibody)

As demonstrated in human meninges, FLT4/VEGFR3 can be successfully detected alongside LYVE1 and MRC1 using either DAB-IHC or fluorescent immunodetection methods , providing reliable identification of lymphatic endothelial cells in complex neural tissues.

What are the critical considerations when using FLT4 antibodies to investigate lymphangiogenesis in tumor microenvironments?

When studying lymphangiogenesis in tumor contexts with FLT4 antibodies, researchers should consider:

Peritumoral vs. intratumoral lymphatics discrimination:

• Use serial sections with H&E staining to correlate vessel location with tumor boundaries

• Combine with basement membrane markers (collagen IV, laminin) to assess vessel integrity

Functional vs. non-functional lymphatic vessel differentiation:

• Perfusion studies with lymphatic-specific tracers

• Assessment of collecting vessel functionality through contraction studies

• Correlation of VEGFR3 expression levels with lymphatic vessel density and functionality

Tumor-associated macrophage interference:
Certain macrophage populations may express VEGFR3, potentially confounding results. Implementation of double immunolabeling with macrophage markers (F4/80, CD68) helps distinguish true lymphatic vessels from macrophages.

Quantification parameters for reliable assessment:

• Lymphatic vessel density (vessels/mm²)

• Lymphatic vessel area (% of tissue area)

• VEGFR3 expression intensity (measured as mean fluorescence intensity)

• Peritumoral vs. intratumoral vessel ratio

Control tissue selection:
Include normal tissue from the same organ to establish baseline VEGFR3 expression patterns and lymphatic vessel characteristics for accurate comparative analysis.

How can FLT4 antibodies be used to elucidate signaling pathway activation in response to lymphangiogenic stimuli?

FLT4/VEGFR3 activates multiple downstream signaling pathways crucial for lymphatic endothelial cell responses. HRP-conjugated antibodies can help investigate these pathways through:

Phosphorylation status assessment:
Using phospho-specific antibodies alongside total FLT4 detection to determine activation state after VEGF-C/D stimulation. Key phosphorylation sites include:

Time-course experiments:

• Stimulate cells with VEGF-C (100-500 ng/ml)

• Collect lysates at multiple time points (5, 15, 30, 60, 120 minutes)

• Blot with HRP-conjugated FLT4 antibodies and phospho-specific antibodies

• Use phosphatase inhibitors in lysis buffer to preserve phosphorylation status

Receptor internalization dynamics:

• Surface biotinylation followed by internalization period

• Immunoprecipitation with FLT4 antibodies

• Western blot with HRP-conjugated FLT4 antibodies

Co-immunoprecipitation studies:
Investigate FLT4 interactions with other signaling components, particularly with respect to heterodimer formation with KDR/VEGFR2 , using HRP-conjugated antibodies for direct detection in pull-down experiments.

What methodological approaches can distinguish between soluble and membrane-bound forms of FLT4/VEGFR3?

FLT4/VEGFR3 exists in both membrane-bound and soluble forms, with the latter potentially functioning as a decoy receptor . Distinguishing between these forms requires specialized approaches:

Differential centrifugation protocol:

• Collect cell culture supernatant or body fluids

• Perform initial low-speed centrifugation (300g, 10 min) to remove cells

• Ultracentrifuge (100,000g, 90 min) to separate membrane vesicles from soluble proteins

• Analyze both fractions by Western blot with HRP-conjugated FLT4 antibodies

Domain-specific antibody selection:

• Use antibodies targeting the extracellular domain (e.g., Tyr25-Ile776) to detect both forms

• Compare with antibodies against the intracellular domain (e.g., aa 1063-1363) to specifically detect membrane-bound forms

Size exclusion approaches:

• Use size-based separation techniques (gel filtration, size exclusion chromatography)

• The soluble form will have lower molecular weight (~75-100 kDa) compared to the membrane-bound form (~150.2 kDa)

Functional assays to distinguish biological activity:

• Competitive binding assays with VEGF-C/D

• Receptor signaling assays in the presence of isolated fractions

• Lymphatic endothelial cell migration/proliferation assays with/without isolated forms

These methodological approaches help researchers accurately distinguish and quantify the different forms of FLT4/VEGFR3, providing insights into their distinct biological roles in normal physiology and disease states.

How can researchers troubleshoot non-specific binding when using HRP-conjugated FLT4 antibodies?

Non-specific binding can significantly compromise experimental results. Researchers can implement several strategies to optimize specificity:

Optimizing blocking conditions:

• Test different blocking agents (BSA, normal serum, commercial blockers)

• Increase blocking time (1-2 hours at room temperature or overnight at 4°C)

• Add 0.1-0.3% Triton X-100 for membrane permeabilization

• Include protein-free blockers if the HRP-conjugated antibody cross-reacts with protein blockers

Antibody dilution optimization:

• Perform serial dilutions beyond the recommended 1:500 (try 1:1000, 1:2000)

• Increase incubation time at higher dilutions to maintain sensitivity while reducing background

Sample-specific considerations:

• For tissues with high endogenous peroxidase activity, quench with H₂O₂ (0.3-3%) before antibody application

• When working with highly vascularized tissues, implement additional washing steps with high-salt PBS (500mM NaCl)

Validation with multiple detection methods:

• Compare results between HRP-conjugated and unconjugated primary antibodies

• Use alternative detection systems (fluorescence) to confirm specificity

• Implement tissue-specific absorption controls to verify binding specificity

What are effective strategies for multiplexing experiments using FLT4 HRP-conjugated antibodies with other markers?

Multiplexing allows simultaneous detection of multiple targets, providing valuable contextual information. When using HRP-conjugated FLT4 antibodies in multiplexed experiments:

Sequential detection protocols:

• Apply HRP-conjugated FLT4 antibody first

• Develop with substrate (preferably precipitating substrates like DAB)

• Thoroughly wash and quench peroxidase activity with H₂O₂ (3%, 10 min)

• Block again and apply second primary antibody

• Use alkaline phosphatase-conjugated secondary antibody with a contrasting substrate (e.g., Vector Blue)

Antibody stripping and reprobing:
For tissues where sequential detection is challenging, implement antibody stripping:

• Glycine buffer (pH 2.2) treatment for 10-15 minutes

• SDS-based stripping buffer (2% SDS, 0.1M β-mercaptoethanol, 62.5mM Tris-HCl)

• Commercial antibody stripping solutions optimized for IHC/IF

Spectral unmixing approaches:
When using fluorescent substrates for HRP:

• Select fluorophores with minimal spectral overlap

• Implement computational spectral unmixing for overlapping emissions

• Include single-stained controls for accurate spectral signatures

Tyramide signal amplification (TSA) multiplexing:
HRP-conjugated antibodies work exceptionally well with TSA:

• Apply HRP-conjugated FLT4 antibody at high dilution (1:1000-1:5000)

• Incubate with tyramide-fluorophore conjugate

• Inactivate HRP with H₂O₂

• Repeat with different HRP-conjugated antibodies and spectrally distinct tyramides

This technique allows for detection of multiple markers even when using antibodies from the same species.

How can FLT4 antibodies contribute to understanding the role of lymphatic vessels in inflammatory diseases?

FLT4/VEGFR3 antibodies are valuable tools for investigating lymphatic involvement in inflammatory conditions:

Lymphatic remodeling assessment in chronic inflammation:

• Quantify lymphatic vessel density and size in inflamed tissues

• Correlate FLT4/VEGFR3 expression levels with inflammatory markers

• Assess lymphatic vessel functionality through tracer uptake studies

Inflammatory cell trafficking analysis:

• Co-stain with immune cell markers (CD45, CD3, CD20) to evaluate perilymphatic immune cell accumulation

• Investigate FLT4/VEGFR3 expression on dendritic cells and macrophages during inflammation

• Analyze chemokine gradient formation along lymphatic vessels using multiplex staining

Intervention assessment protocols:
Evaluate therapeutic interventions targeting lymphangiogenesis with:

• Pre- and post-treatment lymphatic vessel quantification

• FLT4/VEGFR3 phosphorylation status as treatment response indicator

• Correlation with clinical outcomes and biomarkers

Research using FLT4 antibodies has revealed critical insights into meningeal lymphatic vessels, demonstrating co-expression of VEGFR3 with LYVE1 and MRC1 in human brain tissues . This finding highlights the potential role of lymphatic vessels in neuroinflammatory conditions and CNS immune surveillance.

What approaches can validate FLT4 antibody specificity to ensure reliable experimental results?

Rigorous validation is essential for generating trustworthy data with FLT4 antibodies:

Genetic validation approaches:

• Use VEGFR3/FLT4 knockout/knockdown models as negative controls

• Compare staining patterns in tissues with known differential expression

• Implement CRISPR-Cas9 edited cell lines with epitope modifications

Peptide competition assays:

• Pre-incubate antibody with immunizing peptide (for antibodies raised against peptide antigens)

• For recombinant protein immunogens, use the specific protein fragment (e.g., aa 1063-1363 of human FLT4)

• Include both related and unrelated peptides to confirm specificity

Orthogonal detection methods:

• Compare protein detection with mRNA expression (RNA-seq, qRT-PCR)

• Validate with multiple antibodies targeting different epitopes

• Confirm with non-antibody-based methods (e.g., aptamer binding)

Application-specific validation:
For Western blotting:

• Verify the expected molecular weight (~150.2 kDa)

• Include positive control lysates (HUVECs, lymphatic endothelial cells)

• Test in multiple cell types with known expression differences

For immunohistochemistry/immunofluorescence:

• Compare with in situ hybridization patterns

• Include isotype controls at matching concentrations

• Use tissue microarrays containing various tissue types to assess staining patterns