flt4 Antibody

What is FLT4/VEGFR3 and what experimental approaches can be used to study its function?

FLT4, also known as VEGFR3, is a receptor tyrosine kinase primarily involved in lymphangiogenesis and lymphatic system development. It belongs to the protein kinase superfamily and CSF-1/PDGF receptor subfamily . Experimentally, researchers can study FLT4 function through:

• Immunoprecipitation followed by Western blotting to detect phosphorylation status and interaction partners

• Immunofluorescence to visualize cellular localization in lymphatic endothelial cells (LECs)

• Stimulation experiments with VEGF-C (100 ng/ml) to activate the receptor and study downstream signaling

• Gene knockout or knockdown studies to evaluate phenotypic consequences in model systems

FLT4 protein migrates as bands with different molecular weights due to glycosylation: ~175 kDa precursor, ~195 kDa mature form, ~140 kDa non-glycosylated backbone, and a ~125 kDa partially cleaved form .

To ensure antibody specificity for FLT4/VEGFR3:

• Positive controls: Use samples known to express FLT4 such as lymphatic endothelial cells, mouse liver, or mouse spleen tissue

• Negative controls:

  • Compare with FLT4-null mutants or knockdown samples

  • Use isotype control antibodies in parallel experiments

• Cross-reactivity assessment: Check manufacturer data for cross-reactivity with related proteins. For example, some FLT4 antibodies show approximately 15% cross-reactivity with recombinant mouse VEGFR3

• Multiple antibody comparison: Use antibodies from different clones or manufacturers targeting different epitopes of FLT4

• Ligand-stimulation test: Treat cells with VEGF-C and confirm increased phosphorylation of FLT4 using phospho-specific detection methods

The most rigorous validation includes whole mount immunostaining comparing wild-type and flt4-null mutant embryos, as demonstrated in zebrafish models .

How can FLT4 antibodies be used to study the relationship between lymphangiogenesis and cancer progression?

FLT4/VEGFR3 antibodies are valuable tools for investigating lymphangiogenesis in cancer progression through multifaceted approaches:

• Tumor microenvironment analysis:

  • Use immunohistochemistry with FLT4 antibodies (1:50-1:100 dilution) on paraffin-embedded tumor sections to quantify lymphatic vessel density

  • Combine with markers for proliferation (Ki-67) to assess lymphangiogenic activity

• Mechanistic studies:

  • Analyze VEGF-C/VEGFR3 signaling pathway activation using phospho-specific detection methods following immunoprecipitation with FLT4 antibodies

  • Assess PI3K/VEGFR3 complex formation using proximity ligation assays (PLA), which can visualize direct interactions between these proteins in situ

• Treatment response monitoring:

  • Evaluate changes in FLT4 expression and activation following anti-lymphangiogenic therapies

  • Use flow cytometry with APC-conjugated anti-FLT4 antibodies (5 μl per million cells) to monitor circulating endothelial cells expressing VEGFR3

• Metastasis models:

  • Track lymphatic invasion using dual staining with FLT4 and tumor markers

  • Correlate FLT4 expression patterns with metastatic potential and patient outcomes

Recent research demonstrates that TGF-beta family members (TGF-β1, -β2, and -β3) reduce lymphatic marker expression, including VEGFR3, in LECs, providing insight into metastasis regulation mechanisms .

What are the optimal protocols for detecting interactions between FLT4 and its signaling partners?

Detection of FLT4 interactions with signaling partners requires optimized protocols:

• Co-immunoprecipitation (Co-IP):

  • Lyse lymphatic endothelial cells (LECs) in buffer containing protease/phosphatase inhibitors

  • Immunoprecipitate with anti-FLT4 or anti-PI3K antibodies (5-10 μg)

  • Perform Western blotting for detection of interaction partners like PI3K

  • Include stimulation with VEGF-C (100 ng/ml) for 15 minutes to enhance detectable interactions

• Proximity Ligation Assay (PLA):

  • Fix LECs grown in presence or absence of VEGF-C (100 ng/ml)

  • Use antibody pairs (anti-VEGFR3/anti-phospho-PI3K) to detect protein complexes

  • Quantify red spots (indicating interactions) using imaging software

  • Include controls: CD31 staining (green) and DAPI nuclear staining (blue)

• Phosphorylation analysis:

  • After stimulation with VEGF-C or VEGF-A (100 ng/ml), detect phosphorylation using anti-phosphotyrosine antibodies

  • Compare vehicle control, IgG control, and ligand treatments to establish specificity

Research data shows that VEGF-C treatment significantly increases VEGFR3/phospho-PI3K complexes in LECs compared to untreated controls (p<0.01) .

How can researchers reconcile contradictory results from different FLT4 antibody clones?

When faced with contradictory results using different FLT4 antibody clones:

• Epitope mapping assessment:

  • Compare the immunogen sequences of different antibodies. For example, CAB5605 targets amino acids 330-553 of human VEGFR3/FLT4 (NP_002011.2) , while other antibodies may target different regions

  • Consider that differences may be due to detection of specific isoforms or post-translationally modified forms

• Validation in knockout/knockdown systems:

  • Use CRISPR/Cas9 or siRNA to generate FLT4-deficient cells or tissues

  • Test all antibody clones against these negative controls to determine true specificity

• Multiple detection methods:

  • Employ orthogonal techniques (WB, IHC, IF, FC) to confirm findings

  • Consider that certain antibodies perform better in specific applications (e.g., E-3 clone for WB versus D-6 clone for IHC)

• Protein conformation and modification considerations:

  • Note that FLT4 exhibits multiple migration patterns due to glycosylation and proteolytic processing

  • The mature form (~195 kDa) versus cleaved form (~125 kDa) may be preferentially detected by different antibodies

• Cross-species reactivity verification:

  • Test antibodies across multiple species if working with animal models

  • Some antibodies show reactivity with human, mouse, and rat samples, while others are species-specific

What methodological approaches can optimize FLT4 antibody performance in challenging tissue samples?

For optimal FLT4 antibody performance in challenging tissues:

• Antigen retrieval optimization:

  • For paraffin-embedded tissues, test multiple retrieval methods (citrate buffer pH 6.0 vs. EDTA buffer pH 9.0)

  • For cervical squamous metaplasia samples, optimal results have been achieved with overnight incubation at 4°C using 15 μg/mL antibody concentration

• Signal amplification strategies:

  • For low-abundance detection, implement tyramide signal amplification (TSA)

  • In zebrafish models, use anti-rabbit IgG(H+L)-HRP followed by tyramide-Cy3 for enhanced detection

• Background reduction techniques:

  • Include blocking with 5-10% serum from the same species as the secondary antibody

  • Use specific HRP-DAB staining kits (e.g., Anti-Goat HRP-DAB Cell & Tissue Staining Kit) for clean detection

• Multiplexing optimization:

  • When co-staining with other markers (CD31, DAPI), carefully select fluorophores to minimize spectral overlap

  • For co-localization studies, include sequential staining protocols to prevent antibody cross-reactivity

• Species-specific considerations:

  • For zebrafish studies, whole mount immunostaining requires specialized fixation and permeabilization protocols

  • Recommended dilution for zebrafish whole mount staining is 1:500

How can FLT4 antibodies be used to study the effects of TGF-β on lymphatic marker expression?

Research using FLT4 antibodies has revealed important insights into TGF-β regulation of lymphatic markers:

• Experimental design for TGF-β studies:

  • Treat primary human LECs with TGF-β1, -β2, or -β3 at various concentrations (10, 20, or 30 ng/ml)

  • Maintain treatment for extended periods (72-100 hours) to observe sustained effects

  • Use untreated cells as controls

  • Prepare lysates for Western blot analysis using anti-VEGFR3/FLT4 antibodies

• Multi-marker analysis approach:

  • Simultaneously assess multiple lymphatic markers (Lyve-1, Prox-1, VEGFR-3, and vimentin)

  • Use vinculin as a loading control

  • Perform densitometry evaluation using ImageJ software

  • Normalize protein bands to corresponding loading controls

  • Express results as relative expression compared to untreated controls

• Data interpretation guidelines:

  • All three TGF-β isoforms demonstrate dose-dependent suppression of lymphatic markers

  • The effect intensifies with longer exposure (100 hours vs. 72 hours)

  • Correlate VEGFR3/FLT4 reduction with changes in other lymphatic markers to establish pattern of lymphatic phenotype suppression

This methodology has revealed that TGF-β family members significantly downregulate lymphatic marker expression, suggesting a mechanism by which TGF-β might inhibit lymphangiogenesis in pathological conditions.

What are the technical considerations for using FLT4 antibodies in zebrafish research models?

Zebrafish models offer unique advantages for studying FLT4 function, with specific technical considerations:

• Antibody selection and validation:

  • Use rabbit polyclonal antibodies specifically generated against zebrafish Flt4 peptide

  • Verify specificity through comparison between wild-type and flt4-null mutant embryos

  • The epitope sequence CASDELERFEHKHRGA has been validated for zebrafish studies

• Functional validation methods:

  • Test antibodies using Flt4 immunoprecipitates from NIH3T3 cells transfected with plasmids expressing full-length zebrafish Flt4

  • Treat transfected cells with conditioned medium from cells expressing zebrafish Vegfc

  • Confirm activation by Western blotting with antibodies against phosphotyrosine and zebrafish Flt4

• Whole mount immunostaining protocol:

  • Fix embryos at appropriate developmental stages (e.g., 30 hours post-fertilization)

  • Use 1:500 dilution for the primary antibody

  • For signal detection, employ goat anti-rabbit IgG(H+L)-HRP followed by tyramide-Cy3

  • Look for positive staining in the posterior cardinal vein (PCV) endothelial cells in wild-type embryos

• Storage and handling:

  • Store antibody at -80°C

  • Aliquot into small working volumes after initial thaw

  • Use PBS with 0.02% sodium azide as buffer

This approach enables visualization of Flt4 expression patterns during zebrafish development and can be used to study angiogenic processes in vivo.

How can advanced imaging techniques enhance the utility of FLT4 antibodies in lymphatic vessel research?

Advanced imaging techniques significantly expand the research capabilities of FLT4 antibodies:

• Proximity Ligation Assay (PLA) for protein interaction studies:

  • Enables visualization of VEGFR3/phospho-PI3K complexes (red spots) in LECs

  • Allows quantification of protein interactions in response to VEGF-C stimulation

  • Provides spatial information about interaction sites within cells

  • Statistical analysis of PLA signals shows significant increases in VEGFR3/phospho-PI3K complexes after VEGF-C treatment (p<0.01)

• Multi-parameter immunofluorescence:

  • Combine FLT4 antibodies with markers like CD31 (green) and nuclear stain DAPI (blue)

  • Enable simultaneous visualization of lymphatic vessels, blood vessels, and cellular architecture

  • Use specific antibody combinations to distinguish between lymphangiogenic and angiogenic processes

• Super-resolution microscopy applications:

  • Apply techniques like STED or STORM for nanoscale localization of FLT4

  • Investigate receptor clustering and internalization following ligand binding

  • Examine co-localization with signaling partners at sub-diffraction resolution

• Intravital imaging possibilities:

  • Utilize fluorescently-conjugated FLT4 antibodies (FITC, PE, or APC) for in vivo imaging

  • Track lymphatic vessel formation and remodeling in real-time

  • Monitor responses to therapeutic interventions targeting lymphangiogenesis

These advanced techniques provide deeper insights into FLT4 biology than conventional approaches alone, enabling researchers to address complex questions about lymphatic system development and pathology.

What considerations should researchers make when selecting between different conjugated forms of FLT4 antibodies?

Selection between various conjugated forms of FLT4 antibodies requires careful consideration:

• Application-specific selection criteria:

  • For flow cytometry: APC-conjugated antibodies (5 μl per million cells) provide optimal signal-to-noise ratio in the red channel

  • For fluorescence microscopy: FITC-conjugated antibodies work well but may be susceptible to photobleaching

  • For Western blotting: HRP-conjugated antibodies eliminate the need for secondary antibody incubation

  • For immunoprecipitation: Agarose-conjugated antibodies (25% agarose) simplify the pull-down process

• Multiplexing strategy development:

  • When designing multi-color experiments, select FLT4 antibody conjugates with minimal spectral overlap with other fluorophores

  • For three-color imaging, combinations such as FITC-FLT4, PE-conjugated alternative marker, and DAPI are effective

  • Consider Alexa Fluor® conjugates for improved photostability and brightness

• Signal amplification requirements:

  • For low abundance targets, consider using unconjugated primary antibodies followed by amplification steps

  • HRP conjugates can be used with tyramide signal amplification for enhanced sensitivity

  • For direct detection without amplification, brighter fluorophores like PE or Alexa Fluor® conjugates are preferable

• Instrument compatibility verification:

  • Match fluorophore selection to available excitation sources and detection filters

  • APC conjugates require red laser (633 nm) excitation

  • FITC conjugates are compatible with standard blue laser (488 nm) excitation