FBLN5 Antibody, FITC conjugated

What is FBLN5 and why is it significant in biological research?

Fibulin 5 (FBLN5), also known as DANCE, EVEC, or UP50, is a widely expressed extracellular matrix (ECM) glycoprotein that colocalizes with elastic fibers and plays essential roles in proper elastic fiber assembly and vasculogenesis. This protein promotes adhesion of endothelial cells through interaction with integrins via its RGD motif, potentially serving as a vascular ligand for integrin receptors . FBLN5 has garnered research interest due to its involvement in vascular development and remodeling processes . The protein has been implicated in several pathological conditions, including tumor progression and vascular remodeling in atherosclerotic plaques and neointimal thickening following balloon injury . FBLN5's anti-angiogenic properties both in vivo and in vitro make it particularly significant for research into vascular biology and related pathologies .

What are the technical specifications of commercially available FITC-conjugated FBLN5 antibodies?

FITC-conjugated FBLN5 antibodies are typically rabbit polyclonal antibodies with immunoglobulin G (IgG) isotype. These antibodies target various epitopes of the Fibulin 5 protein, with different products recognizing specific amino acid sequences. For example, some antibodies target amino acids 70-82, while others recognize regions 126-206/448 . The antibodies are generally supplied at concentrations around 1 μg/μl or 1 mg/ml in liquid format . They typically show reactivity against human FBLN5, with many also cross-reacting with mouse and rat FBLN5 . These antibodies are supplied in storage buffers containing components such as glycerol (50%), PBS, and preservatives like sodium azide or ProClin 300 .

What are the recommended applications for FITC-conjugated FBLN5 antibodies?

FITC-conjugated FBLN5 antibodies are versatile tools suitable for multiple experimental applications. The primary applications include:

• Immunofluorescence (IF/ICC): These antibodies are particularly useful for immunofluorescence applications with recommended dilutions ranging from 1:50 to 1:200 . The FITC conjugation eliminates the need for secondary antibody incubation, simplifying the protocol and reducing background.

• Western Blotting (WB): Some FITC-conjugated FBLN5 antibodies can be used for western blotting at dilutions between 1:500 and 1:3000 . Researchers should be aware that the FITC conjugation may affect sensitivity in this application.

• ELISA: These antibodies can be utilized in ELISA protocols at approximately 1:1000 dilution . The fluorescent properties of FITC can be leveraged for detection in certain ELISA formats.

• Immunohistochemistry (IHC): Some FITC-conjugated FBLN5 antibodies are validated for IHC applications, allowing visualization of FBLN5 in tissue sections .

When designing experiments, researchers should optimize antibody concentrations for their specific experimental conditions, tissue types, and detection systems .

What are the proper storage and handling conditions for FITC-conjugated FBLN5 antibodies?

Proper storage and handling of FITC-conjugated FBLN5 antibodies is critical for maintaining their functionality and fluorescence intensity. These antibodies should be stored in light-protected vials or covered with light-protecting material (such as aluminum foil) to prevent photobleaching of the FITC fluorophore . For short-term storage, the antibodies are stable for at least 12 months when kept at 4°C . For longer storage (up to 24 months), the antibodies can be diluted with up to 50% glycerol and stored at -20°C to -80°C .

Researchers should minimize freeze-thaw cycles as repeated freezing and thawing can compromise both enzyme activity and antibody binding properties . Many commercial FITC-conjugated FBLN5 antibodies are already supplied in a buffer containing 50% glycerol, which helps maintain stability during freezing . Some products recommend storing at 4°C for 12 months in their provided aqueous buffered solutions containing BSA, glycerol, and sodium azide .

How can FITC-conjugated FBLN5 antibodies be used to investigate hypoxia-induced changes in endothelial cells?

FITC-conjugated FBLN5 antibodies provide a powerful tool for investigating hypoxia-induced changes in endothelial cells. Research has shown that FBLN5 is upregulated by hypoxia in endothelial cells through a HIF-1 dependent mechanism . To study this phenomenon:

• Experimental Setup: Researchers can culture endothelial cells (such as HUVEC) under normoxic and hypoxic conditions (typically 1-2% O₂) for various time periods. Maximal FBLN5 induction occurs after approximately 24 hours of hypoxia exposure .

• Visualization Protocol: After exposure to hypoxia, cells can be fixed with 4% paraformaldehyde. For extracellular FBLN5 detection, researchers should avoid permeabilization steps; for total FBLN5, cells should be permeabilized with 0.5% Tween 20 . The FITC-conjugated FBLN5 antibody can then be applied directly (typically at 1:50-1:200 dilution) .

• Co-staining Approach: For comprehensive analysis, nuclei can be counterstained with Hoechst 33342 and actin fibers with phalloidin conjugated to a spectrally distinct fluorophore (e.g., Alexa Fluor 633) .

• Pathway Analysis: To investigate the signaling mechanisms involved, researchers can combine this visualization with inhibitors of the PI3K/Akt/mTOR pathway (such as LY294002 and rapamycin), which block hypoxia-induced FBLN5 upregulation . Similarly, dimethyl oxalylglycine (DMOG), which prevents HIF-1α degradation, can be used to mimic hypoxic conditions .

• Quantification Methods: Confocal microscopy images can be quantitatively analyzed to measure changes in FBLN5 expression, comparing intracellular versus extracellular localization under different conditions .

This methodological approach allows researchers to investigate not only FBLN5 expression changes but also its potential role in endothelial cell survival during hypoxic stress .

What optimization strategies are recommended for multi-color immunofluorescence using FITC-conjugated FBLN5 antibodies?

When designing multi-color immunofluorescence experiments using FITC-conjugated FBLN5 antibodies, several optimization strategies should be considered:

• Spectral Compatibility: FITC has excitation/emission maxima around 495/519 nm. When selecting additional fluorophores, choose those with minimal spectral overlap, such as DAPI (358/461 nm) for nuclei, Cy3 (550/570 nm) or Texas Red (589/615 nm) for other targets .

• Sequential Imaging: If using confocal microscopy with fluorophores that have partial spectral overlap, employ sequential scanning rather than simultaneous acquisition to minimize bleed-through artifacts.

• Antibody Concentration Titration: Perform titration experiments to determine the optimal concentration of FITC-conjugated FBLN5 antibody. Start with the manufacturer's recommended range (1:50-1:200) and adjust based on signal-to-noise ratio .

• Fixation Protocol Optimization: For extracellular matrix proteins like FBLN5, the fixation method significantly impacts epitope accessibility. Compare 4% paraformaldehyde with methanol fixation to determine which preserves the structural context while maintaining antibody binding sites .

• Autofluorescence Control: Include unstained controls to assess tissue autofluorescence, particularly important when working with tissues rich in elastin and collagen, which naturally fluoresce in the FITC spectrum.

• Signal Amplification Considerations: For tissues with low FBLN5 expression, consider using a biotin-streptavidin system with FITC-conjugated streptavidin for signal amplification, rather than direct FITC-conjugated antibody detection.

• Cross-Reactivity Testing: When combining with other antibodies, perform single-staining controls to ensure no cross-reactivity exists between antibodies, particularly if using multiple rabbit-derived antibodies.

What are the key considerations for validating FITC-conjugated FBLN5 antibody specificity?

Validating the specificity of FITC-conjugated FBLN5 antibodies is crucial for generating reliable and reproducible research data. Researchers should implement the following validation approaches:

• Positive and Negative Controls: Use tissues or cell lines with known FBLN5 expression levels as positive controls. Human vascular tissues, which naturally express FBLN5, make excellent positive controls . Conversely, FBLN5 knockout models or siRNA-treated cells serve as optimal negative controls .

• Peptide Competition Assay: Pre-incubate the FITC-conjugated FBLN5 antibody with excess immunizing peptide (if available from the manufacturer) before application to samples. Specific staining should be substantially reduced or eliminated.

• Western Blot Correlation: Confirm that the antibody detects a protein of the expected molecular weight (~50 kDa for FBLN5) in western blot analysis of the same samples used for immunofluorescence .

• Comparison Across Species: When using antibodies claimed to be cross-reactive among species (human, mouse, rat), verify that staining patterns are consistent with known FBLN5 distribution in each species .

• Knockdown Verification: Use FBLN5 siRNA or shRNA to reduce expression in cultured cells, then confirm reduced staining intensity correlates with reduced protein levels by western blot .

• Alternative Antibody Comparison: Compare staining patterns with other FBLN5 antibodies that recognize different epitopes. Concordant staining patterns increase confidence in specificity.

• Isotype Control: Include an isotype-matched control antibody conjugated to FITC to assess non-specific binding due to the antibody class rather than antigen specificity.

What are the common issues encountered with FITC-conjugated FBLN5 antibodies and their solutions?

Researchers working with FITC-conjugated FBLN5 antibodies may encounter several technical challenges. Here are common issues and their methodological solutions:

When troubleshooting FITC-conjugated FBLN5 antibody applications, systematically adjust one parameter at a time while maintaining careful documentation of all experimental conditions.

How can FITC-conjugated FBLN5 antibodies be optimized for quantitative analysis of FBLN5 expression?

Quantitative analysis of FBLN5 expression using FITC-conjugated antibodies requires careful optimization to ensure accurate and reproducible measurements:

• Standard Curve Calibration: Create a standard curve using recombinant FBLN5 protein at known concentrations to relate fluorescence intensity to protein amount. This is particularly useful for flow cytometry or quantitative ELISA applications.

• Image Acquisition Standardization: For microscopy-based quantification, maintain consistent acquisition parameters:

  • Use identical exposure times, gain, and offset settings across all samples

  • Capture images at a bit depth sufficient to distinguish intensity differences (12-16 bit recommended)

  • Include fluorescence standards in each imaging session to normalize between experiments

• Background Correction Methodology: Implement rigorous background subtraction:

  • Measure and subtract local background for each image

  • Use rolling ball algorithms with radius larger than typical cell size

  • Include isotype control samples to determine non-specific binding contribution

• Signal Normalization Approaches: Normalize FBLN5 signal:

  • To cell number using nuclear counterstains

  • To cell area using membrane or cytoskeletal markers

  • To a housekeeping protein that remains stable under experimental conditions

• Dynamic Range Optimization: Ensure measurements fall within the linear range of detection:

  • Avoid pixel saturation by checking histogram during acquisition

  • Establish lower detection limit by measuring signal-to-noise ratio

  • Consider using high-dynamic-range imaging techniques for samples with wide expression variations

• Batch Effect Mitigation: Control for variations between experimental batches:

  • Process all comparative samples simultaneously when possible

  • Include internal reference samples across batches

  • Apply statistical normalization methods appropriate for fluorescence data

• Segmentation Strategy: For image-based quantification, develop consistent segmentation protocols:

  • Define clear criteria for identifying positive regions

  • Use automated thresholding methods (Otsu, Li, etc.) for objectivity

  • Implement watershed algorithms for distinguishing adjacent positive regions

How can FITC-conjugated FBLN5 antibodies contribute to understanding vascular pathologies?

FITC-conjugated FBLN5 antibodies can significantly advance our understanding of vascular pathologies through multiple methodological approaches:

• Atherosclerosis Research: FBLN5 has been linked to vascular remodeling in atherosclerotic plaques . Researchers can use FITC-conjugated FBLN5 antibodies to:

  • Visualize FBLN5 distribution within plaque regions compared to healthy vessel walls

  • Correlate FBLN5 expression with markers of endothelial dysfunction

  • Track changes in FBLN5 localization during plaque progression in animal models

• Angiogenesis Studies: Given FBLN5's anti-angiogenic properties , these antibodies can help:

  • Identify differential expression of FBLN5 in tumor vasculature versus normal vessels

  • Monitor FBLN5 levels during therapeutic interventions targeting angiogenesis

  • Assess FBLN5 interaction with endothelial integrins via co-localization studies with integrin markers

• Hypoxia Response Mechanisms: Since FBLN5 is upregulated by hypoxia in endothelial cells through HIF-1 dependent mechanisms , researchers can:

  • Compare FBLN5 levels in ischemic tissues using dual staining with hypoxia markers like pimonidazole

  • Investigate temporal relationships between HIF-1α stabilization and FBLN5 expression

  • Assess the protective role of FBLN5 against hypoxia-induced apoptosis in vessel walls

• Elastic Fiber Disorders: As FBLN5 is essential for proper elastic fiber assembly , these antibodies allow:

  • Assessment of FBLN5 distribution in tissues from patients with connective tissue disorders

  • Evaluation of therapeutic approaches aimed at restoring normal elastogenesis

  • Visualization of structural relationships between FBLN5 and other elastic fiber components

• Vascular Injury Models: In neointimal thickening following vascular injury , these antibodies enable:

  • Temporal mapping of FBLN5 expression during vessel wall remodeling

  • Correlation between FBLN5 levels and smooth muscle cell proliferation/migration

  • Evaluation of potential therapeutic interventions targeting FBLN5-related pathways

These applications collectively provide comprehensive insights into the role of FBLN5 in vascular health and disease, potentially identifying new therapeutic targets for vascular pathologies.

What protocols are recommended for studying FBLN5 in cell-matrix interactions using FITC-conjugated antibodies?

For investigating FBLN5's role in cell-matrix interactions, the following optimized protocols utilizing FITC-conjugated FBLN5 antibodies are recommended:

Protocol 1: Co-localization of FBLN5 with ECM Components

• Sample Preparation:

  • Culture cells on glass coverslips coated with relevant matrix proteins (collagen, elastin, fibronectin)

  • For tissue sections, use 5-8 μm unfixed frozen sections or paraffin sections with appropriate antigen retrieval

• Fixation and Processing:

  • For extracellular matrix visualization: Fix with 4% paraformaldehyde (10 min, RT) without permeabilization

  • For total FBLN5 visualization: After fixation, permeabilize with 0.5% Tween 20 (5 min, RT)

• Blocking:

  • Block with 5% normal goat serum in PBS (1 hour, RT)

• Antibody Incubation:

  • Apply FITC-conjugated FBLN5 antibody (1:100 dilution) together with unconjugated primary antibodies against ECM components of interest (overnight, 4°C)

  • Wash 3x with PBS

  • Apply spectrally compatible secondary antibodies for the unconjugated primaries (1 hour, RT)

• Counterstaining:

  • Nuclei: Hoechst 33342 (1:5000, 10 min)

  • Actin cytoskeleton: Alexa Fluor 633-phalloidin (1:40, 20 min)

• Mounting and Imaging:

  • Mount with anti-fade medium

  • Image using confocal microscopy with sequential scanning to prevent bleed-through

  • Analyze co-localization using Pearson's or Mander's coefficients

Protocol 2: FBLN5-Integrin Binding Assay

• Cell Preparation:

  • Culture endothelial cells expressing integrins known to interact with FBLN5 (αvβ3, αvβ5, α9β1)

  • Prepare comparison groups: untreated, RGD peptide-treated (competitive inhibition), and integrin-blocking antibody treated

• FBLN5 Binding Visualization:

  • Incubate live cells with recombinant FBLN5 protein (5-10 μg/ml, 1 hour, 37°C)

  • Wash gently with warm PBS

  • Fix with 2% paraformaldehyde (mild fixation to preserve surface binding)

  • Apply FITC-conjugated FBLN5 antibody (1:100, 1 hour, RT)

• Integrin Co-staining:

  • Apply antibodies against relevant integrins conjugated to compatible fluorophores (e.g., Cy3)

• Analysis:

  • Quantify FBLN5 binding in control vs. treated conditions

  • Assess co-localization with integrins

  • Correlate binding with functional assays (adhesion, migration)

These protocols enable detailed investigation of FBLN5's interactions with extracellular matrix components and cell surface receptors, providing insights into its functional roles in tissue development and homeostasis.

What emerging applications of FITC-conjugated FBLN5 antibodies show promise for advancing vascular biology research?

Emerging applications of FITC-conjugated FBLN5 antibodies show significant potential for advancing vascular biology research through innovative methodological approaches:

• Live-Cell Imaging of FBLN5 Dynamics: Development of non-toxic, membrane-permeable FITC-conjugated FBLN5 antibody fragments (Fab or nanobodies) could enable real-time visualization of FBLN5 secretion and matrix incorporation in living endothelial cells. This would provide unprecedented insights into the dynamic regulation of elastic fiber assembly.

• Super-Resolution Microscopy Applications: Combining FITC-conjugated FBLN5 antibodies with super-resolution techniques like STORM or STED microscopy could reveal nanoscale organization of FBLN5 within elastic fibers, potentially identifying structural patterns previously undetectable with conventional microscopy.

• Multiplexed Tissue Analysis: Integration of FITC-conjugated FBLN5 antibodies into multiplexed immunofluorescence panels with simultaneous detection of 10+ markers could map complex relationships between FBLN5 expression and vascular cell phenotypes in health and disease. This approach would be particularly valuable for analyzing heterogeneous vascular tissues.

• Microfluidic Organ-on-Chip Models: Application of FITC-conjugated FBLN5 antibodies in microfluidic vascular models could facilitate assessment of flow-dependent FBLN5 expression and matrix deposition under precisely controlled hemodynamic conditions, bridging the gap between static cell culture and in vivo complexity.

• Single-Cell Proteomics Correlation: Combining FACS using FITC-conjugated FBLN5 antibodies with single-cell proteomics could identify cell populations with varying FBLN5 expression levels and correlate these with broader proteomic signatures, potentially uncovering new regulatory networks.

• Intravital Microscopy: Development of in vivo compatible FITC-conjugated FBLN5 antibodies could enable real-time tracking of FBLN5 dynamics during vascular remodeling events in living organisms using intravital microscopy techniques.

• Biomaterial Functionalization: FITC-conjugated FBLN5 antibodies could be employed to assess the incorporation of FBLN5 into tissue-engineered vascular grafts, potentially guiding the development of biomaterials that better mimic natural vessel properties.

These emerging applications represent frontier areas where FITC-conjugated FBLN5 antibodies could significantly expand our understanding of vascular biology and potentially inform therapeutic strategies for vascular diseases.