Fn1 Antibody, FITC conjugated

What is Fibronectin (Fn1) and what cellular functions does it perform?

Fibronectin (Fn1) is a high-molecular-weight glycoprotein found in the extracellular matrix (ECM) that plays critical roles in multiple cellular processes. It functions as a ligand that binds cell surfaces and various compounds including collagen, fibrin, heparin, DNA, and actin. Fibronectin is fundamentally involved in cell adhesion, cell motility, opsonization, wound healing, and maintenance of cell shape. Additionally, it participates in osteoblast compaction through the fibronectin fibrillogenesis cell-mediated matrix assembly process, which is essential for osteoblast mineralization. Recent research has identified its role in regulating type I collagen deposition by osteoblasts and acting as a ligand for the LILRB4 receptor, inhibiting FCGR1A/CD64-mediated monocyte activation .

How does FITC conjugation enhance Fn1 antibody functionality in research applications?

FITC conjugation to Fn1 antibodies enables direct visualization of fibronectin within cells and tissues without requiring secondary antibody incubation steps. This conjugation provides several methodological advantages:

• Reduces experimental time by eliminating secondary antibody incubation

• Minimizes background signal from non-specific secondary antibody binding

• Allows for multicolor imaging when combined with other fluorophore-conjugated antibodies

• Enables live-cell imaging applications when using membrane-permeable variants

• Produces consistent signal intensity between experiments

For optimal results in immunofluorescence applications, FITC-conjugated Fn1 antibodies are typically used at dilutions between 1:50 and 1:200, depending on the specific antibody formulation and experimental conditions .

What aspects of fibronectin structure and organization have been revealed through FITC-conjugated antibody imaging?

Recent advanced imaging studies using FITC-conjugated antibodies have dramatically revised our understanding of fibronectin's structural organization. Contrary to the traditional view of fibronectin fibrils as continuous fibers with extended, periodically aligned molecules, single-molecule localization microscopy and live imaging have revealed that:

• Fibronectin fibrils are composed of roughly spherical nanodomains containing six to eleven Fn1 dimers

• These nanodomains become organized into linear arrays with an average periodicity of 105±17 nm

• Fibronectin fibrillogenesis initiates at the cell periphery as distinct, brightly fluorescent densities

• These Fn1 densities move centripetally and align into linear arrays of "beads"

• The beaded architecture is consistent regardless of the antibody epitope used for detection

This nanodomain organization has been observed in cell culture as well as within tissues, suggesting it represents the fundamental structural unit of fibronectin fibrils .

What controls should be included when using Fn1 antibody, FITC conjugated for immunofluorescence studies?

When designing immunofluorescence studies using FITC-conjugated Fn1 antibodies, researchers should implement the following controls:

Additionally, when studying fibronectin nanodomain structures, researchers should validate findings using multiple antibodies recognizing distinct Fn1 epitopes. As demonstrated in recent research, the beaded appearance of fibronectin fibrils is consistent regardless of the antibody used, including polyclonal antibodies recognizing multiple Fn1 epitopes and monoclonal antibodies targeting specific regions .

How can researchers optimize fixation protocols to preserve Fn1 epitopes for FITC-conjugated antibody detection?

Optimization of fixation protocols is crucial for preserving fibronectin structure while maintaining epitope accessibility. Based on experimental evidence:

• Paraformaldehyde fixation (4%): Provides good structural preservation while maintaining most epitopes. Optimal fixation time is typically 10-15 minutes at room temperature.

• Methanol fixation: Useful for certain epitopes but can disrupt some protein-protein interactions. For fibronectin studies focused on nanodomain organization, this may alter the native architecture.

• Dual fixation: Sequential paraformaldehyde followed by methanol can provide better preservation of both structure and epitope accessibility.

• Gentle permeabilization: Use 0.1-0.2% Triton X-100 for 5-10 minutes after paraformaldehyde fixation to facilitate antibody access while preserving fibronectin fibril organization.

• Antigen retrieval: If necessary, mild heat-mediated antigen retrieval (citrate buffer, pH 6.0) can improve epitope accessibility without disrupting fibril structure.

For studies specifically examining fibronectin nanodomain arrangements, researchers should verify that their fixation protocol preserves the characteristic beaded structure through comparison with live-cell imaging of Fn1-FP fusion proteins .

How should researchers address photobleaching when using FITC-conjugated Fn1 antibodies in prolonged imaging sessions?

FITC is relatively prone to photobleaching compared to other fluorophores, which can limit imaging duration. Researchers can employ several strategies to mitigate this issue:

• Anti-fade mounting media: Use specialized mounting media containing anti-photobleaching agents compatible with FITC.

• Oxygen scavenging systems: Include oxygen scavengers in imaging buffer to reduce photobleaching rates.

• Image acquisition parameters: Reduce exposure time, excitation intensity, and frequency of imaging to minimize cumulative light exposure.

• Computational correction: Apply photobleaching correction algorithms during post-processing of time-lapse data.

• Alternative strategy: For extended live-cell imaging studies of fibronectin dynamics, consider using genetic approaches with Fn1-FP fusion proteins as demonstrated in recent studies. These CRISPR/Cas9 knock-in strategies have proven effective for visualizing fibronectin fibrillogenesis over time without antibody application .

How can Fn1 antibody, FITC conjugated be used to investigate fibronectin fibrillogenesis mechanisms?

FITC-conjugated Fn1 antibodies provide powerful tools for investigating fibronectin fibrillogenesis through several advanced approaches:

• Time-course immunofluorescence: Fixed-cell imaging at progressive time points can capture different stages of fibril assembly.

• Co-localization studies: FITC-Fn1 antibodies can be combined with antibodies against integrins, other ECM components, or cytoskeletal proteins to investigate molecular associations during fibrillogenesis.

• Super-resolution microscopy: Techniques such as STORM, PALM, or STED using FITC-conjugated antibodies can resolve nanoscale features of fibronectin organization.

• Correlative approaches: Combine FITC-Fn1 immunofluorescence with electron microscopy to correlate optical and ultrastructural information.

Recent research has revealed that fibronectin fibrillogenesis begins at the cell periphery with distinct fluorescent densities that move centripetally in parallel with F-actin, eventually aligning into linear arrays of "beads." These studies have shown co-localization of higher intensity fibronectin with integrin α5β1 in both non-fibrillar and fibrillar adhesions, suggesting coordinated assembly mechanisms .

What insights have been gained about fibronectin nanodomain organization using advanced imaging techniques?

Advanced imaging techniques have revolutionized our understanding of fibronectin's structural organization:

• Nanodomain composition: Single-molecule localization microscopy has revealed that fibronectin fibrils are composed of roughly spherical nanodomains containing approximately 6-11 Fn1 dimers.

• Spatial arrangement: These nanodomains organize into linear arrays with a consistent periodicity of 105±17 nm.

• Stability characteristics: Fibronectin nanodomain arrays remain resistant to deoxycholate treatment and maintain their periodicity even in the absence of cells, suggesting inherent structural stability.

• Conservation of structure: The periodic nanodomain structure has been observed across different experimental settings, including between cells in culture and within tissues.

• Epitope independence: The nanodomain periodicity remains constant regardless of which antibody epitope is targeted, confirming this represents genuine structural organization rather than epitope accessibility artifacts .

How can researchers utilize Fn1 antibody, FITC conjugated in combination with other techniques to study fibronectin-integrin interactions?

To comprehensively study fibronectin-integrin interactions, researchers can combine FITC-conjugated Fn1 antibodies with complementary techniques:

• Dual immunofluorescence: Co-stain for fibronectin (using FITC-Fn1) and integrin receptors (using differently conjugated antibodies) to visualize spatial relationships.

• FRET analysis: Use FITC-Fn1 antibodies with acceptor fluorophore-conjugated integrin antibodies to detect molecular proximity through Förster resonance energy transfer.

• Proximity ligation assay (PLA): Combine FITC-Fn1 antibodies with secondary antibodies for PLA to detect and quantify specific protein-protein interactions.

• Live-cell imaging: For dynamic studies, combine FITC-Fn1 antibody staining with integrin-specific probes in time-lapse microscopy.

• Biochemical cross-linking: Complement imaging data with cross-linking experiments to identify molecular interactions.

Recent research has demonstrated that domains of higher fluorescence intensity of fibronectin co-localize with areas of higher intensity of integrin α5β1 in both non-fibrillar and fibrillar adhesions, with both showing characteristic "beaded" architecture in fibrillar adhesions .

What are common causes of weak signal when using Fn1 antibody, FITC conjugated in immunofluorescence experiments?

When encountering weak signal with FITC-conjugated Fn1 antibodies, researchers should consider:

Additionally, researchers should verify antibody quality through known positive controls. For Fn1 antibodies, fibroblast cultures typically show robust fibronectin expression and are useful as positive controls .

How should researchers interpret punctate versus fibrillar patterns when visualizing fibronectin with FITC-conjugated antibodies?

The pattern of fibronectin staining provides important biological information:

• Punctate/dotted pattern: Recent research has demonstrated that the "beaded" or dotted appearance of fibronectin fibrils is not an artifact but represents the true nanodomain organization of fibronectin. These roughly spherical nanodomains contain multiple Fn1 dimers and are organized into linear arrays with regular spacing .

• Fibrillar pattern: At lower magnification or resolution, these nanodomain arrays appear as continuous fibrils. The alignment of nanodomains creates the fibrillar appearance observed in conventional microscopy.

• Diffuse/non-fibrillar pattern: Often represents newly secreted, non-assembled fibronectin or denatured protein due to sample processing issues.

• Mixed patterns: Different stages of fibril assembly may be present in the same sample, especially during active ECM remodeling or in experimental manipulation of fibril formation.

Researchers should be aware that the dotted appearance of Fn1 fibrils in tissues and cells, as revealed by recent high-resolution imaging, reflects the genuine structural organization of fibronectin into nanodomain arrays rather than technical artifacts .

What approaches can validate questionable results obtained using Fn1 antibody, FITC conjugated?

When results obtained with FITC-conjugated Fn1 antibodies are ambiguous, researchers should implement validation strategies:

• Multiple antibody validation: Use several antibodies recognizing different fibronectin epitopes to confirm staining patterns. Recent research has shown that the beaded appearance of Fn1 fibrils is consistent regardless of the antibody used, including polyclonal antibodies recognizing multiple epitopes along the Fn1 molecule .

• Alternative detection methods: Compare results with non-FITC detection systems or unconjugated primary antibodies with fluorophore-conjugated secondaries.

• Genetic approaches: When available, compare antibody staining with genetically encoded fluorescent protein fusions (Fn1-FP). Recent work has demonstrated that CRISPR/Cas9 knock-in strategies can generate viable cell lines and embryos expressing Fn1-mEGFP, Fn1-mScarlet-I, Fn1-Neon Green, or Fn1-tdTomato fusion proteins that accurately report fibronectin localization .

• Functional validation: Perform deoxycholate (DOC) insolubility assays to biochemically verify the incorporation of Fn1 into the ECM, which correlates with proper fibril formation .

• Complementary techniques: Validate observations using orthogonal methods such as Western blotting, ELISA, or mass spectrometry.

How are super-resolution microscopy techniques enhancing research using Fn1 antibody, FITC conjugated?

Super-resolution microscopy has transformed our understanding of fibronectin structure and organization:

• Single-molecule localization microscopy (SMLM): Has revealed that Fn1 fibrils are composed of roughly spherical nanodomains rather than continuous fibers of extended, periodically aligned molecules. This technique has demonstrated that these nanodomains contain approximately 6-11 Fn1 dimers and are arranged with consistent 105±17 nm periodicity .

• Structured illumination microscopy (SIM): Provides enhanced resolution that can resolve fibronectin nanodomain organization in intact tissue samples and thicker specimens where SMLM might be challenging.

• Stimulated emission depletion (STED) microscopy: Offers high-resolution imaging of fibronectin organization without the complex reconstruction algorithms required for SMLM.

• Expansion microscopy: Physical expansion of specimens combined with conventional fluorescence microscopy can achieve super-resolution imaging of fibronectin structures with standard FITC-conjugated antibodies.

• Correlative light and electron microscopy (CLEM): Combines the molecular specificity of FITC-conjugated Fn1 antibodies with the ultrastructural context provided by electron microscopy.

These advanced techniques have fundamentally changed our understanding of fibronectin architecture, revealing that the traditional view of continuous fibrils is incomplete .

How can multiplex imaging with Fn1 antibody, FITC conjugated reveal new aspects of ECM-cell interactions?

Multiplex imaging strategies combining FITC-conjugated Fn1 antibodies with other markers can provide comprehensive insights into ECM-cell interactions:

• Multi-color immunofluorescence: Simultaneously visualize fibronectin, other ECM components, cellular receptors, and signaling molecules.

• ECM tension sensors: Combine FITC-Fn1 staining with FRET-based tension sensors to correlate fibronectin organization with mechanical forces.

• Live-cell multiplexing: Use FITC-Fn1 antibody fragments with live cell markers to study dynamic interactions between fibronectin assembly and cellular processes.

• Cyclic immunofluorescence: Sequential staining and imaging rounds allow visualization of dozens of markers on the same sample, providing comprehensive interaction maps.

• Spatial transcriptomics integration: Correlate fibronectin organization with gene expression patterns in the same tissue section.

Recent research has demonstrated that higher intensity domains of fibronectin co-localize with areas of higher intensity integrin α5β1 in both non-fibrillar and fibrillar adhesions. This co-localization reveals important insights into how cells engage with and assemble the fibronectin matrix through specific receptor interactions .

What role does Fn1 antibody, FITC conjugated play in investigating fibronectin's involvement in disease processes?

FITC-conjugated Fn1 antibodies are valuable tools for investigating fibronectin's roles in various pathological conditions:

• Cancer research: Investigate how fibronectin organization influences tumor cell invasion and metastasis. Recent research has shown that anastellin (a fibronectin fragment) and superfibronectin (a fibronectin polymer) inhibit tumor growth, angiogenesis, and metastasis through mechanisms involving p38 MAPK activation and inhibition of lysophospholipid signaling .

• Fibrosis studies: Examine how fibronectin fibrillogenesis contributes to pathological matrix deposition in liver, lung, kidney, and cardiac fibrosis.

• Wound healing disorders: Investigate abnormalities in fibronectin organization and deposition in impaired wound healing conditions.

• Developmental disorders: Study how disruptions in fibronectin nanodomain organization affect embryonic development, as fibronectin's beaded structure has been observed in embryonic tissues such as pharyngeal arches and heart .

• Metabolic disorders: Explore fibronectin's role in metabolic regulation, as it has been shown that fibronectin secreted by contracting muscle induces liver autophagy and systemic insulin sensitization via hepatic ITGA5:ITGB1 integrin receptor signaling .