FBN2 Antibody, FITC conjugated

What is the biological function of FBN2 and why is it an important research target?

FBN2 belongs to the fibrillin family of extracellular glycoproteins that form the structural backbone of 10-12 nm calcium-binding microfibrils in connective tissues. These microfibrils can exist either in association with elastin or in elastin-free bundles. FBN2-containing microfibrils specifically regulate the early process of elastic fiber assembly in tissues .

From a pathological perspective, mutations in the FBN2 gene resulting in impaired assembly of fibrillin-2 can lead to congenital contractural arachnodactyly (CCA), also known as Beals syndrome or distal arthrogryposis . Additionally, mutations in the 8-cysteine motif of Fibrillin-2 alter its binding to microfibril-associated glycoprotein-1 (MAGP-1), potentially increasing the severity of CCA . This connection to human disease makes FBN2 a significant target for both basic and translational research.

What experimental applications are suitable for FBN2 Antibody, FITC conjugated?

The FITC-conjugated FBN2 antibody is particularly valuable for fluorescence-based applications:

• Immunofluorescence (IF) microscopy: Recommended dilutions typically range from 1:50-1:200 for immunohistochemistry-paraffin (IHC-P) applications .

• Flow cytometry: The FITC conjugation enables direct detection without secondary antibodies.

• Immunocytochemistry (ICC): For cellular localization studies.

• Fluorescence-based ELISA: When direct detection is preferred.

The FITC conjugation provides direct visualization without requiring secondary antibody incubation steps, which can be advantageous for:

How does the FITC conjugation affect epitope binding and detection sensitivity compared to unconjugated FBN2 antibodies?

FITC conjugation can potentially impact antibody performance in several ways:

For applications requiring maximum sensitivity, researchers might consider using unconjugated FBN2 antibody with highly sensitive secondary detection systems, while direct FITC conjugation offers workflow advantages and reduced cross-reactivity concerns .

What are the optimal fixation and permeabilization methods when using FITC-conjugated FBN2 antibody for immunofluorescence studies?

Since FBN2 is an extracellular matrix protein that constitutes the backbone of microfibrils inserting into basement membranes , fixation and permeabilization protocols should be optimized to preserve extracellular structures while allowing antibody access:

• Recommended fixation protocols:

  • 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature

  • Avoid methanol fixation which can disrupt epitope structure

  • For tissue sections, antigen retrieval may be necessary using TE buffer pH 9.0 or citrate buffer pH 6.0

• Permeabilization considerations:

  • Mild detergents (0.1-0.3% Triton X-100) should be used cautiously as FBN2 is primarily extracellular

  • For cultured cells, consider limited permeabilization (0.1% Triton X-100 for 5-10 minutes)

  • For some applications, permeabilization may be unnecessary since FBN2 is secreted and deposited into the extracellular matrix in a nonfibrillar form by epithelial cells

• Blocking recommendations:

  • 5-10% normal serum (species different from host of primary antibody)

  • 1-3% BSA in PBS

  • Include 0.1% Tween-20 to reduce non-specific binding

These parameters should be empirically optimized for each experimental system and tissue type.

What is the recommended protocol for dual immunofluorescence labeling with FITC-conjugated FBN2 antibody and other ECM protein markers?

Dual immunofluorescence allows for visualization of FBN2 in relation to other extracellular matrix components. Due to FBN2's role in microfibril formation and elastic fiber assembly, co-localization studies with elastin, fibrillin-1, and other ECM proteins are common.

Recommended protocol:

• Sample preparation:

  • Fix samples with 4% PFA for 15-20 minutes

  • Perform antigen retrieval if using paraffin sections (TE buffer pH 9.0 recommended)

  • Block with 10% normal serum + 1% BSA in PBS for 1 hour

• Primary antibody incubation:

  • For FITC-conjugated FBN2 antibody: Use at 1:50-1:200 dilution

  • For unconjugated second primary antibody: Follow manufacturer's recommendation

  • Incubate overnight at 4°C in blocking buffer

• Secondary antibody incubation (for the unconjugated primary only):

  • Select a secondary antibody with a fluorophore spectrally distinct from FITC (e.g., Cy3, Alexa Fluor 594)

  • Incubate for 1-2 hours at room temperature

  • Wash thoroughly to remove unbound antibodies

• Counterstaining and mounting:

  • Counterstain nuclei with DAPI

  • Use antifade mounting medium to preserve FITC signal

  • Consider adding 10 mM HEPES buffer (pH 8.0) to mounting medium to optimize FITC fluorescence

• Imaging considerations:

  • Acquire FITC signal using excitation ~495 nm, emission ~519 nm

  • Acquire non-overlapping channels sequentially to prevent bleed-through

  • Include single-stained controls to confirm specificity

This approach enables visualization of FBN2 in relation to other ECM components to better understand microfibril assembly and interactions.

How can researchers quantify FBN2 expression levels using FITC-conjugated antibodies in tissue samples?

Quantification of FBN2 expression using FITC-conjugated antibodies requires careful experimental design and image analysis:

When publishing, report all image acquisition parameters, threshold determination methods, and quantification approaches to ensure reproducibility .

How does FBN2 antibody staining pattern differ between normal tissues and those affected by congenital contractural arachnodactyly (CCA)?

Congenital contractural arachnodactyly (CCA), also known as Beals syndrome, results from mutations in the FBN2 gene. When comparing normal versus CCA-affected tissues using FBN2 antibody staining:

When analyzing such tissues, researchers should consider both qualitative assessment of staining patterns and quantitative measurements of signal distribution and intensity. Correlation with electron microscopy findings can provide additional structural context to immunofluorescence observations.

What are the methodological approaches for studying FBN2 expression during embryonic development using FITC-conjugated antibodies?

Studying FBN2 expression during embryonic development presents unique challenges and opportunities:

• Developmental time points:

  • FBN2 expression precedes FBN1, making early developmental stages particularly important

  • Key time points include early organogenesis, cardiovascular system formation, and limb development

• Sample preparation considerations:

  • Optimize fixation to preserve delicate embryonic tissues (typically 2-4% PFA)

  • Consider whole-mount immunofluorescence for early embryos

  • Serial sectioning with consistent orientation for comparative analyses

  • Careful antigen retrieval to avoid tissue damage

• 3D visualization techniques:

  • Confocal z-stacks with 3D reconstruction

  • Light sheet microscopy for whole embryos

  • Tissue clearing methods (CLARITY, CUBIC, etc.) for deeper tissue imaging

• Co-labeling strategies:

  • Developmental markers specific to each stage

  • Lineage-specific markers

  • Extracellular matrix proteins (elastin, fibrillin-1)

  • Cell proliferation and differentiation markers

• Quantitative developmental mapping:

  • Spatiotemporal expression mapping

  • Correlation with tissue morphogenesis

  • Comparison with gene expression data

• Controls and validation:

  • Stage-matched controls

  • Multiple embryos per developmental stage

  • Validation with in situ hybridization for FBN2 mRNA

  • Correlation with known developmental phenotypes in FBN2 mutant models

This comprehensive approach allows researchers to understand the dynamic expression patterns of FBN2 during development and its relationship to tissue morphogenesis and ECM assembly .

What are common problems encountered when using FITC-conjugated FBN2 antibodies, and how can they be addressed?

When working with FITC-conjugated FBN2 antibodies, researchers may encounter several challenges:

• High background fluorescence:

  • Cause: Insufficient blocking, non-specific binding, or tissue autofluorescence

  • Solution:

    • Increase blocking time/concentration (use 5-10% normal serum + 1-3% BSA)

    • Add 0.1-0.3% Triton X-100 to blocking buffer

    • Include autofluorescence reduction steps (e.g., 0.1% Sudan Black B treatment)

    • Optimize antibody concentration (titrate from 1:50 to 1:200)

• Weak or absent signal:

  • Cause: Insufficient antigen retrieval, epitope masking, or low antibody concentration

  • Solution:

    • Optimize antigen retrieval (try TE buffer pH 9.0 as recommended, or alternatively citrate buffer pH 6.0)

    • Increase antibody concentration

    • Extend incubation time (up to 48 hours at 4°C)

    • Use signal amplification systems compatible with FITC

• Photobleaching:

  • Cause: FITC's moderate photostability

  • Solution:

    • Use anti-fade mounting media containing radical scavengers

    • Reduce exposure during imaging

    • Capture FITC channel first in multi-channel imaging

    • Consider alternative conjugates with higher photostability

• Non-specific binding:

  • Cause: Cross-reactivity with similar proteins

  • Solution:

    • Include additional blocking proteins

    • Pre-absorb antibody with non-specific proteins

    • Include appropriate negative controls

    • Validate with alternative FBN2 antibodies

• Variable results across experiments:

  • Cause: Inconsistent protocol execution or lot-to-lot antibody variation

  • Solution:

    • Standardize all protocol steps

    • Document lot numbers and prepare large batches of working dilutions

    • Include positive controls in each experiment

    • Consider purchasing larger antibody quantities of the same lot

These troubleshooting approaches should be systematically tested and documented to establish optimal conditions for specific experimental systems .

How do different tissue preparation methods affect FBN2 epitope preservation and FITC signal intensity?

The choice of tissue preparation method significantly impacts both FBN2 epitope preservation and FITC signal quality:

Optimization recommendations:

• For paraffin-embedded tissues:

  • Use antigen retrieval with TE buffer pH 9.0 as recommended for FBN2 antibodies

  • Alternative: citrate buffer pH 6.0 retrieval if TE buffer is ineffective

  • Retrieval time should be empirically determined (typically 10-20 minutes)

• For fresh frozen tissues:

  • Rapid fixation in 4% PFA (10 minutes) prior to antibody incubation

  • Gentle permeabilization to maintain extracellular matrix structure

• For cultured cells:

  • 4% PFA fixation for 10-15 minutes at room temperature

  • Mild permeabilization (0.1% Triton X-100 for 5 minutes)

• General considerations:

  • FITC signal is optimal at slightly alkaline pH (7.5-8.0)

  • Avoid prolonged exposure to light during all processing steps

  • Include antioxidants in buffers when possible to preserve FITC fluorescence

When working with challenging tissues or samples, preliminary optimization experiments comparing different preparation methods are strongly recommended .