FBLN2 Antibody, FITC conjugated

What is the optimal protocol for FBLN2 antibody immunofluorescence staining in tissue sections?

Optimal immunofluorescence protocols for FBLN2 detection should follow these methodological steps:

• Prepare 5 μm tissue sections (FFPE or frozen)

• Perform antigen retrieval using 1 mM EDTA buffer (pH 8) under high pressure

• Block sections with 2.5% goat serum for 20-30 minutes at room temperature

• Incubate with primary FBLN2 antibody at 1:10,000-1:15,000 dilution for 30 minutes

• Wash three times with PBS

• Apply Alexa Fluor conjugated secondary antibody (if using unconjugated primary) at 1:1,000 dilution for 20 minutes in darkness

• Mount using an antifade reagent containing DAPI

• Image using confocal microscopy with a 63× oil immersion lens

For FITC-conjugated FBLN2 antibodies, skip steps 5-6 and proceed directly to mounting after primary antibody incubation, adjusting exposure settings to account for FITC's excitation maximum at 495 nm and emission maximum at 525 nm .

How should researchers quantify FBLN2 expression in different tissue compartments?

Quantification of FBLN2 expression requires a systematic approach:

• Establish a scoring system based on staining intensity and distribution (0=negative to 3=strong)

• Assess staining separately in different tissue compartments (epithelium, basement membrane, stroma)

• For immunohistochemistry: Score DAB staining intensity visually or using image analysis software

• For immunofluorescence: Measure fluorescence intensity using software like ImageJ

• Include multiple fields (at least 10 per sample) to account for tissue heterogeneity

• Use consistent exposure settings across all samples

• Normalize to appropriate housekeeping proteins or internal controls

• Compare with other ECM markers such as COLIV, POSTN, TN-C for comprehensive analysis

Research by Rizwan et al. demonstrated effective scoring of FBLN2 expression in 65 breast tumors using this approach, correlating expression patterns with other ECM components .

What are critical controls when using FBLN2 antibody, FITC conjugated in experimental protocols?

Rigorous experimental design requires the following controls:

Essential Controls:

• Negative control: Primary antibody omission to assess background autofluorescence

• Isotype control: Non-specific IgG (rabbit IgG for polyclonal FBLN2 antibodies) at matching concentration

• Positive control: Tissue known to express FBLN2 (e.g., mammary gland TEB regions, developing cardiac valves)

• Blocking peptide control: Pre-incubation of antibody with recombinant FBLN2 protein (301-440AA region)

• Fluorophore control: Unconjugated primary + secondary detection to compare with direct FITC conjugation

Additional Validation Approaches:

• siRNA knockdown validation in cell models

• Comparison with antibodies targeting different FBLN2 epitopes

• Cross-validation with RNA expression data where available

• Western blot confirmation of specificity at expected 126.6 kDa (with potential isoforms) .

How can researchers optimize the fluorescein/protein (F/P) ratio for FITC-conjugated FBLN2 antibodies?

Optimization of F/P ratio is critical for balancing sensitivity and specificity:

• The optimal F/P ratio for antibodies is typically between 2:1 and 6:1

• Test different FITC to antibody molar ratios during conjugation:

  • 5:1 ratio typically results in F/P of 1-2

  • 10:1 ratio typically results in F/P of 2-4

  • 20:1 ratio typically results in F/P of 3-6

• Avoid over-labeling (F/P > 6) which causes:

  • Increased non-specific binding

  • Decreased quantum yield through self-quenching

  • Altered antibody specificity

• For initial optimization, perform small-scale conjugations at different ratios before scaling up

• Purify conjugated antibody using gel filtration to remove unconjugated FITC .

How can FBLN2 antibody, FITC conjugated be utilized to investigate FBLN2-Notch2 interactions in biological systems?

Investigation of FBLN2-Notch2 interactions requires specialized protocols:

• Co-localization studies:

  • Use FITC-conjugated FBLN2 antibody alongside Notch2 antibody (with spectrally distinct fluorophore)

  • Analyze co-localization using confocal microscopy and quantify using Pearson's correlation coefficient

• Proximity ligation assay (PLA):

  • Combine FBLN2 and Notch2 antibodies with oligonucleotide-conjugated secondary antibodies

  • Proximity (<40 nm) generates fluorescent signal through rolling circle amplification

  • Quantify interaction events per cell

• Immunoprecipitation validation:

  • Perform IP with anti-FBLN2 antibody followed by Notch2 detection

  • Technical approach: Transfect cells with 3×Flag-tagged FBLN2, immunoprecipitate with anti-3×Flag or anti-Notch2 antibodies, and detect using respective antibodies

  • High-affinity binding between Fibulin2 (Ala28–Leu1184) and Notch2 (Leu26–Gln530) has been demonstrated with KD = 1.22 × 10^-9 ± 3.16 × 10^-11

• FRET analysis:

  • For live-cell studies, utilize FITC-conjugated FBLN2 antibody with Notch2 antibody conjugated to a FRET-compatible acceptor

  • Measure energy transfer as indicator of molecular proximity .

What methodological approaches should be used when studying FBLN2's role in extracellular matrix organization using FITC-conjugated antibodies?

Study of FBLN2's ECM functions requires multi-dimensional approaches:

• Co-immunostaining protocols:

  • Combine FBLN2-FITC antibody with antibodies against known binding partners:

    • Basement membrane: COLIV, laminin (LN111, LN332)

    • Elastic fibers: Fibrillin-1, elastin

    • Matrix proteins: Fibronectin, versican

  • Use spectral unmixing to distinguish FITC from tissue autofluorescence

• Temporal analysis of ECM assembly:

  • Time-course studies during development or wound healing

  • FBLN2 is strongly associated with pubertal and early pregnant mouse mammary epithelial outgrowth

  • Correlate with basement membrane formation and integrity

• Functional manipulation experiments:

  • Compare ECM organization in normal vs. FBLN2-depleted conditions

  • Measure FBLN2 interaction with at least 14 known binding partners:

    • 12 ECM proteins

    • 2 membrane receptors (integrin β3/ITGB3 and integrin α5/ITGA5)

• Adhesion assays:

  • Quantify cell adhesion to laminin-coated surfaces with/without FBLN2

  • FBLN2 increases adhesiveness of mammary cell lines to ECM-coated surfaces through its RGD binding site .

How can researchers prevent photobleaching when working with FBLN2 antibody, FITC conjugated?

FITC is particularly susceptible to photobleaching, requiring specific preventive measures:

• Sample preparation optimization:

  • Use high-quality antifade mounting media containing anti-photobleaching agents

  • ProLong® Gold antifade reagent with DAPI is effective for FBLN2 immunofluorescence

  • Store slides at -20°C in darkness when not being imaged

• Imaging parameters:

  • Minimize exposure time and laser/light intensity

  • Use neutral density filters to reduce excitation light

  • Employ frame averaging rather than increased intensity for better signal-to-noise ratio

  • Consider deconvolution to enhance weak signals post-acquisition

• Alternative approaches:

  • Consider sequential imaging of multiple fields rather than extended exposure of a single field

  • For time-lapse studies, use time intervals that allow signal recovery

  • If extensive imaging is required, consider antibodies with more photostable fluorophores (Alexa Fluor conjugates) or indirect immunofluorescence

• Storage considerations:

  • Store FITC-conjugated antibodies at -20°C or -80°C

  • Avoid repeated freeze-thaw cycles

  • Protect from light with amber vials or aluminum foil .

What methodological solutions exist for distinguishing specific from non-specific binding when using FBLN2 antibody, FITC conjugated?

Differentiating specific from non-specific signals requires systematic approach:

• Optimization of blocking conditions:

  • Test different blocking agents: serum (2.5% goat or horse serum), BSA (1-5%), commercial blockers

  • Extend blocking time from standard 20-30 minutes to 1-2 hours for high background samples

  • Include 0.1-0.3% Triton X-100 or 0.05% Tween-20 in blocking solution for improved penetration

• Antibody titration:

  • Test dilution series (starting from 1:5,000 to 1:20,000 for FBLN2 antibodies)

  • Plot signal-to-noise ratio against antibody concentration to identify optimal dilution

  • Consider the F/P ratio when determining optimal concentration (higher F/P requires lower antibody concentration)

• Signal validation approaches:

  • Competitive inhibition with purified FBLN2 protein (301-440AA region)

  • Compare staining pattern with mRNA expression (in situ hybridization)

  • Cross-validate with multiple FBLN2 antibodies recognizing different epitopes

  • Genetic models: compare wild-type versus FBLN2-knockout tissues

• Technical considerations:

  • Include stringent wash steps (3× PBS washes of 5 minutes each)

  • For tissues with high autofluorescence, consider tissue autofluorescence quenching methods .

What are the methodological considerations when using FBLN2 antibody, FITC conjugated to study cancer progression?

FBLN2's role in cancer progression requires specialized experimental approaches:

• Tissue microarray analysis protocol:

  • Compare FBLN2 expression across tumor stages and normal tissue

  • Score based on staining intensity (0-3 scale) in different compartments

  • Correlate with clinical parameters and other markers

• Cell invasion assays:

  • Use FBLN2-FITC antibody to track FBLN2 distribution during invasion

  • Combine with markers of epithelial-mesenchymal transition (EMT)

  • Co-stain with markers that have been correlated with FBLN2 in cancer:

    • Vimentin (Vim)

    • Smooth muscle actin (SMA)

    • Tenascin-C (TN-C)

    • Periostin (POSTN)

• Functional studies:

  • Establish FBLN2 knockdown and overexpression cell models using lentiviral vectors

  • Compare migration, invasion, and metastatic potential

• Clinical correlation:

  • Assess FBLN2 in liquid biopsies (serum/plasma)

  • Study has suggested FBLN2 contributes to metastasis and invasion in lung adenocarcinoma and hepatocellular carcinoma .

How can researchers validate changes in FBLN2 expression and localization during developmental processes?

Developmental biology studies of FBLN2 require specific methodological approaches:

• Temporal sampling strategy:

  • Collect tissues at defined developmental stages

  • For mammary gland: pubertal, pregnancy, lactation, and involution phases

  • FBLN2 is strongly associated with pubertal and early pregnant mouse mammary epithelial outgrowth

• Co-localization analysis:

  • Use triple immunofluorescence with FBLN2-FITC plus:

    • Basement membrane markers (laminin, collagen IV)

    • Cell type markers (epithelial, myoepithelial, stromal)

  • Analyze using confocal microscopy with Z-stack acquisition

  • Quantify spatial relationships using 3D reconstruction

• Functional correlation:

  • Study binding partners of FBLN2 during development

  • FBLN2 has at least 14 known binding partners:

    • 12 ECM proteins

    • 2 membrane receptors (integrin β3 and integrin α5)

  • Assess basement membrane integrity with/without FBLN2

  • Compare with knockout models when available

• Technical considerations:

  • Use Alexa Fluor conjugated secondary antibodies (1:1,000 dilution)

  • Image with confocal microscopy (63× oil immersion recommended)

  • For co-localization studies, ensure proper channel separation and controls .

What are the recommended methods for quantitative analysis of FBLN2 distribution patterns in tissue sections using FITC-conjugated antibodies?

Quantitative analysis requires sophisticated image processing approaches:

• Image acquisition protocol:

  • Use consistent exposure settings across all samples

  • Capture multiple representative fields (minimum 10)

  • Include Z-stack acquisition (0.5-1μm steps) for 3D analysis

  • Record both FITC channel and autofluorescence channel for background correction

• Computational analysis approaches:

  • Segment tissue compartments (epithelium, stroma, basement membrane)

  • Measure fluorescence intensity in each compartment

  • Quantify linear vs. diffuse staining patterns

  • Calculate relative distribution patterns normalized to total FBLN2 signal

• Advanced metrics:

  • Co-localization coefficients with basement membrane markers

  • Spatial correlation with binding partners

  • Distance mapping from cellular boundaries

  • Pattern recognition for fibrillar vs. punctate distribution

• Statistical analysis:

  • Use non-parametric tests for scoring data (Mann-Whitney, Kruskal-Wallis)

  • Apply multivariate analysis to correlate with other markers

  • Implement machine learning approaches for pattern recognition .

How does the binding affinity of FBLN2 antibodies impact experimental design considerations in protein interaction studies?

Binding affinity considerations are crucial for interaction studies:

• Quantitative assessment methods:

  • Measure antibody-antigen affinity using surface plasmon resonance (SPR)

  • Determine KD values to ensure high-affinity detection

  • For FBLN2-Notch2 interaction studies, note the high binding affinity:

    • Fibulin2 (Ala28–Leu1184) displayed high binding affinity for Notch2 (Leu26–Gln530)

    • KD = 1.22 × 10^-9 ± 3.16 × 10^-11

• Experimental design implications:

  • For high-affinity interactions (nanomolar KD), use lower antibody concentrations

  • For weaker interactions, consider crosslinking approaches

  • Account for potential epitope masking in protein complexes

  • Use gentle washing conditions to preserve weaker interactions

• Advanced interaction analysis:

  • Consider biolayer interferometry (BLI)-based biosensor technology

  • Perform gradient experiments with proportional dilutions (25, 50, and 100 nM)

  • Use co-immunoprecipitation with 3×Flag-tagged FBLN2 to validate interactions

  • Include proper washing and elution conditions based on affinity measurements .