VEGFB Antibody, FITC conjugated

What is VEGFB and how does it differ from other VEGF family members?

VEGFB (Vascular Endothelial Growth Factor B) is a member of the VEGF family that plays a distinct role in angiogenesis compared to its homolog VEGF-A. Recent research has revealed that VEGFB functions as an endogenous inhibitor of angiogenesis by inhibiting the FGF2/FGFR1 pathway, particularly when the latter is abundantly expressed. Unlike VEGF-A, which primarily promotes angiogenesis, VEGF-B has been shown to prevent excessive angiogenesis by binding to FGFR1, inducing FGFR1/VEGFR1 complex formation, and suppressing FGF2-induced Erk activation . This unexpected anti-angiogenic function suggests caution is needed when modulating VEGF-B activity for treating neovascular diseases.

VEGF-B binds primarily to VEGFR1 (Vascular Endothelial Growth Factor Receptor-1) and has been associated with metabolic homeostasis and neuroprotection. Notably, in breast cancer patients, low VEGF-B levels correlate with higher cancer risks, while high levels are associated with lower risks .

What are the key specifications of VEGFB Antibody, FITC conjugated for research use?

VEGFB Antibody, FITC conjugated typically shares similar specifications to other VEGF family antibodies. Based on comparable products, researchers should expect:

For optimal experimental outcomes, researchers should verify the exact specifications of their particular antibody lot, as subtle variations may exist between manufacturers .

What are the recommended storage and handling conditions for maximizing antibody stability?

FITC-conjugated antibodies require special handling to maintain their fluorescent properties. Store the antibody in light-protected vials or cover with a light-protecting material (e.g., aluminum foil) to prevent photobleaching . Long-term stability is achieved through the following measures:

For short-term storage (up to 12 months), keep the antibody at 4°C in the dark. For extended storage (up to 24 months), dilute with up to 50% glycerol and store at -20°C to -80°C . Avoid repeated freeze-thaw cycles as they compromise both enzyme activity and antibody binding capacity. Aliquoting the antibody upon receipt is strongly recommended to minimize freeze-thaw damage .

Some preparations may be lyophilized, requiring reconstitution before use. Typically, reconstitution is performed in PBS (pH 7.4) containing protein stabilizers such as BSA (≈1-20 mg/ml), and preservatives like sodium azide (0.02%) or Proclin300 (0.03%) .

What are the optimal application-specific dilutions and methodological considerations?

Dilution requirements vary depending on the specific application and experimental context:

Always perform titration experiments to determine the optimal concentration for your specific experimental conditions. Signal-to-noise ratio should guide final dilution selection .

How can researchers design experiments to investigate VEGF-B's interaction with the FGF2/FGFR1 pathway?

To investigate VEGF-B's role in inhibiting the FGF2/FGFR1 pathway, researchers can design multi-faceted experimental approaches:

• Proximity Ligation Assays (PLA): This technique directly visualizes VEGFR1/FGFR1 complex formation in cells. Treat samples with VEGF-B and compare to controls (such as PlGF treatment), followed by detection using specific antibodies against FGFR1 and VEGFR1. Increased red dots would indicate complex formation, as demonstrated in previous research .

• Co-immunoprecipitation Studies: Use immunoprecipitation with anti-FGFR1 antibodies followed by Western blotting with anti-VEGFR1 antibodies (or vice versa) to quantify VEGFR1/FGFR1 complex formation in response to VEGF-B treatment .

• Competition Binding Assays: Employ Surface Plasmon Resonance (SPR) or competitive ELISA to assess whether VEGF-B competes with FGF2 for FGFR1 binding. This can help determine the binding kinetics and potential inhibitory mechanisms .

• Angiogenesis Assays: Compare FGF2-induced angiogenesis with and without VEGF-B treatment using endothelial tube formation assays, sprouting assays, or in vivo models to functionally validate VEGF-B's inhibitory effects .

When using FITC-conjugated VEGFB antibody in these contexts, it serves primarily as a detection tool to visualize VEGF-B localization and potentially its co-localization with receptors.

What controls should be included when using VEGFB Antibody, FITC conjugated in immunofluorescence studies?

A robust experimental design requires multiple controls to ensure valid interpretation of results:

• Negative Controls:

  • Isotype control: Use a FITC-conjugated IgG from the same host species (rabbit) at the same concentration to assess non-specific binding

  • Secondary antibody only (if using indirect detection methods)

  • Unstained samples to establish baseline autofluorescence

  • VEGFR1-knockout or VEGF-B-knockout tissues/cells when available

• Positive Controls:

  • Tissues known to express VEGF-B (heart, lung, brain as indicated in search result )

  • Recombinant VEGF-B-treated cell lines with validated expression

• Blocking Controls:

  • Pre-incubation with unconjugated anti-VEGF-B to demonstrate specificity

  • Peptide competition using the immunizing peptide to confirm epitope specificity

• Multiplexing Controls:

  • When performing multi-color immunofluorescence, include single-color controls to assess spectral overlap

  • Use VEGF-A antibodies in parallel to distinguish between family members

For proximity ligation assays investigating VEGFR1/FGFR1 interactions, crucial controls include using each antibody alone, as demonstrated in previous research where this approach yielded no complex formation signals .

How can researchers minimize photobleaching when working with FITC-conjugated antibodies?

FITC is particularly susceptible to photobleaching compared to some other fluorophores. To maximize signal retention:

• Sample Preparation:

  • Add anti-fade reagents to mounting media (e.g., n-propyl gallate, p-phenylenediamine, or commercial anti-fade solutions)

  • Consider using ProLong Gold or similar mounting media containing anti-fade compounds

  • Store slides at 4°C in complete darkness when not imaging

• Microscopy Practices:

  • Minimize exposure time during imaging

  • Reduce excitation light intensity to the minimum needed for adequate signal

  • Use neutral density filters when appropriate

  • Capture FITC channel images first in multi-channel experiments

  • Utilize confocal microscopy with controlled laser power rather than widefield fluorescence when possible

• Advanced Approaches:

  • Consider photobleaching correction algorithms during image analysis

  • For quantitative studies, include fluorescent standards to normalize signal intensity between samples

  • For extended time-lapse imaging, consider alternative conjugates with greater photostability than FITC

These steps are particularly important when investigating subtle co-localization patterns or when quantitative analysis is required.

How can researchers distinguish between VEGF-A and VEGF-B signals in complex tissue samples?

Distinguishing between closely related VEGF family members requires careful experimental design:

• Antibody Selection:

  • Verify that the VEGF-B antibody's immunogen is from a region that differs from VEGF-A

  • Review the antibody's cross-reactivity data in the datasheet; ideal antibodies show no cross-reactivity with VEGF-A

  • For VEGF-B, antibodies targeting the C-terminal region may provide better specificity, as this region contains sequences distinct from VEGF-A

• Experimental Validation:

  • Perform Western blot analysis to confirm detection of the correct molecular weight (VEGF-B: ~21-27 kDa depending on isoform; VEGF-A: ~21-45 kDa depending on isoform)

  • Use recombinant VEGF-A and VEGF-B proteins as positive controls in parallel experiments

  • Consider siRNA knockdown of VEGF-B to confirm signal specificity

• Co-staining Approaches:

  • Perform dual immunofluorescence with different fluorophore-conjugated antibodies specific to VEGF-A and VEGF-B

  • Analyze co-localization patterns quantitatively using appropriate software (e.g., JACoP plugin for ImageJ)

  • Look for differential expression patterns, as VEGF-A and VEGF-B often show distinct tissue distribution

• Functional Validation:

  • Incorporate functional assays that distinguish between VEGF-A's pro-angiogenic effects and VEGF-B's newly discovered anti-angiogenic properties, particularly in FGF2-rich environments

What are common causes of non-specific binding or high background when using VEGFB Antibody, FITC conjugated?

High background or non-specific binding can compromise data quality. Key troubleshooting approaches include:

• Common Causes and Solutions:

• FITC-Specific Considerations:

  • FITC has a relatively broad emission spectrum, which can overlap with tissue autofluorescence

  • Consider alternative mounting media specifically designed to reduce autofluorescence

  • For tissues with high endogenous fluorescence (e.g., brain, liver), include an autofluorescence quenching step

• Validation Approaches:

  • Compare staining patterns between multiple antibodies targeting different epitopes of VEGF-B

  • Include absorption controls where the antibody is pre-incubated with excess antigen

How has recent research changed our understanding of VEGF-B's role in angiogenesis?

Recent findings have dramatically altered our understanding of VEGF-B's role in angiogenesis and have significant implications for therapeutic approaches:

Mechanistically, researchers have discovered that:

• VEGF-B binds directly to FGFR1 with a KD value of approximately 17 nM

• VEGF-B induces VEGFR1/FGFR1 complex formation in multiple cell types and tissues

• VEGF-B competes with FGF2 for FGFR1 binding

• VEGF-B inhibits FGF2-induced Erk activation

• VEGF-B demonstrates anti-angiogenic effects in multiple in vitro and in vivo assays

The binding site for FGFR1 has been mapped to the C-terminus of VEGF-B, specifically to regions covered by synthetic peptides 10 and 11, which do not overlap with amino acid residues important for VEGFR1 binding. This suggests the possibility of simultaneous binding of VEGF-B to both FGFR1 and VEGFR1 .

These findings raise important questions about current therapeutic approaches that inhibit VEGF-B along with other VEGF family members for treating neovascular diseases, as they might inadvertently promote angiogenesis in FGF2-rich environments.

What techniques can researchers use to investigate VEGFR1/FGFR1 complex formation in relation to VEGF-B?

Researchers have several sophisticated techniques at their disposal to investigate the newly discovered VEGFR1/FGFR1 complex formation:

• In Situ Proximity Ligation Assay:

  • This technique directly visualizes protein-protein interactions in intact cells

  • In previous studies, treatment of human retinal endothelial cells (HRECs) with VEGF-B increased the association of FGFR1 with VEGFR1, while PlGF treatment showed no effect

  • The method requires antibodies specific to each protein (VEGFR1 and FGFR1) and yields red dots where the proteins are in close proximity

• Co-immunoprecipitation (Co-IP):

  • Research has shown that VEGFR1 co-immunoprecipitates with FGFR1 in mouse tissues including brain, lung, and heart, demonstrating the existence of naturally occurring endogenous VEGFR1/FGFR1 complexes

  • Intravitreal injection of VEGF-B increased the amount of VEGFR1/FGFR1 complex in mouse retinae

• Surface Plasmon Resonance (SPR) Analysis:

  • SPR analysis using recombinant VEGFR1/FGFR1 heterodimer proteins has shown that VEGF-B binds to these heterodimers with KD values of 1.4-3.7 nM

  • Notably, these binding affinities are higher than those for the FGFR1 homodimer (17 nM), suggesting that VEGF-B may preferably bind to the VEGFR1/FGFR1 heterodimer

• Heterodimer Protein Engineering:

  • Researchers have generated soluble VEGFR1/FGFR1 heterodimer recombinant proteins containing the extracellular domains of both receptors connected by a linker

  • These engineered proteins serve as valuable tools for studying binding interactions and could be utilized in various binding assays

FITC-conjugated VEGF-B antibodies can be particularly valuable in visualizing the localization of VEGF-B in relation to these receptor complexes using immunofluorescence microscopy.

What are the implications of VEGF-B's dual nature for cancer research and therapeutic development?

The discovery of VEGF-B's anti-angiogenic properties has profound implications for cancer research and therapeutics:

• Prognostic Indicator:

  • In breast cancer patients, low VEGF-B levels have been associated with higher cancer risks, while high levels correlate with lower risks

  • This contradicts the traditional view that all VEGF family members promote tumor progression through angiogenesis

• Therapeutic Reconsideration:

  • Current anti-angiogenic therapies that inhibit multiple VEGF family members, including VEGF-B, may have context-dependent effects

  • In tumors with high FGF2/FGFR1 expression, inhibiting VEGF-B might potentially promote rather than inhibit angiogenesis

  • This could help explain why anti-VEGF therapies have failed to show benefits in many types of cancers

• Context-Dependent Function:

  • VEGF-B's effect appears to depend on the local FGF2/FGFR1 levels

  • Targeting VEGF-B might require personalized approaches based on tumor microenvironment analysis

• Novel Therapeutic Strategies:

  • Understanding the VEGF-B binding sites for FGFR1 (specifically peptides 10 and 11 covering the C-terminus) could lead to the development of peptide mimetics that selectively inhibit FGF2-induced angiogenesis

  • The VEGFR1/FGFR1 heterodimer presents a novel therapeutic target distinct from targeting either receptor alone

• Diagnostic Applications:

  • FITC-conjugated VEGF-B antibodies could be valuable for assessing VEGF-B levels in patient samples

  • The ratio of VEGF-B to FGF2 might serve as a predictive biomarker for response to anti-angiogenic therapies

These findings underscore the need for a more nuanced approach to anti-angiogenic therapy in cancer, with careful consideration of the specific molecular context of each tumor.