RBFOX2 Antibody, FITC conjugated

What is RBFOX2 and what cellular functions does it perform?

RBFOX2 (RNA binding protein fox-1 homolog 2) is a multifunctional RNA binding protein that primarily regulates alternative splicing events by binding to 5'-UGCAUGU-3' elements within pre-mRNA transcripts. RBFOX2 prevents binding of U2AF2 to the 3'-splice site, thereby modulating RNA processing outcomes. Beyond alternative splicing regulation, RBFOX2 also influences mRNA stability and translation efficiency, making it a critical post-transcriptional regulator . RBFOX2 has been identified as playing important roles in neurogenesis, heart development, and other tissue-specific developmental processes . In some contexts, RBFOX2 can act as a coregulatory factor of estrogen receptor alpha (ER-alpha) and, together with RNA binding proteins like RBPMS and MBNL1/2, activates vascular smooth muscle cells alternative splicing events .

In which tissue types and cellular compartments is RBFOX2 typically expressed?

RBFOX2 shows tissue-specific and developmentally regulated expression patterns. In neural tissues, RBFOX2 is expressed in retinal ganglion cells (RGCs) of all subtypes, horizontal cells, and GABAergic amacrine cells (ACs), including cholinergic starburst ACs and NPY (neuropeptide Y) and EBF1 (early B-cell factor 1)-positive ACs . Unlike its family member RBFOX1, which changes subcellular localization during development, RBFOX2 maintains a predominantly nuclear localization throughout retinal development, with expression detected as early as embryonic day 12 (E12) . In cardiac tissue, RBFOX2 plays crucial roles in embryonic heart development, influencing cardiac chamber formation and outflow tract development . The consistent nuclear localization of RBFOX2 correlates with its primary role in splicing regulation within the nucleus.

What are the critical storage and handling considerations for maintaining RBFOX2 Antibody activity?

To maintain optimal activity of RBFOX2 Antibody, FITC conjugated, store the antibody at -20°C or -80°C immediately upon receipt . The product is typically shipped at 4°C but requires lower storage temperatures for long-term preservation of activity. Avoid repeated freeze-thaw cycles as these can significantly reduce antibody performance through protein denaturation and fluorophore degradation . The antibody is supplied in a buffer containing 0.03% Proclin 300 (preservative), 50% Glycerol, and 0.01M PBS at pH 7.4, which helps maintain stability during storage . When working with the antibody, aliquot into smaller volumes before freezing to minimize freeze-thaw cycles, and protect from prolonged light exposure to prevent photobleaching of the FITC fluorophore. Always centrifuge the product briefly before opening to collect all liquid at the bottom of the vial after thawing.

How should I design immunostaining protocols using RBFOX2 Antibody, FITC conjugated for tissue sections?

When designing immunostaining protocols for tissue sections using RBFOX2 Antibody, FITC conjugated, follow this methodological approach:

• Fixation and Processing: Use 4% paraformaldehyde fixation for 14-16 hours at 4°C, followed by paraffin embedding or cryopreservation depending on the tissue type and experimental requirements.

• Antigen Retrieval: For paraffin sections, perform heat-induced epitope retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0). For cryosections, a milder antigen retrieval may be sufficient.

• Blocking: Block nonspecific binding with 5-10% normal serum (from the same species as your secondary antibody if using additional primary antibodies) in PBS containing 0.1-0.3% Triton X-100 for permeabilization, for 1-2 hours at room temperature.

• Primary Antibody Incubation: Dilute RBFOX2 Antibody, FITC conjugated at 1:50 to 1:200 (optimize for your specific tissue) in blocking buffer and incubate for 14-16 hours at room temperature in a humidifying chamber .

• Washing: Perform 4-5 washes with PBS containing 0.1% Triton X-100, 5-10 minutes each .

• Nuclear Counterstaining: Incubate with DAPI (4′,6-diamino-2-phenylindole dihydrochloride) or TO-PRO-3 for 30 minutes at room temperature in the dark .

• Mounting and Imaging: Mount slides with anti-fade mounting medium and image using appropriate filters for FITC (excitation ~495 nm, emission ~519 nm) and counterstains.

When performing co-staining with other antibodies, ensure spectral compatibility with FITC and modify the protocol to include appropriate sequential or simultaneous staining steps depending on the host species of additional primary antibodies.

What controls should I include when validating RBFOX2 Antibody, FITC conjugated for my specific application?

To properly validate RBFOX2 Antibody, FITC conjugated for your specific application, implement these essential controls:

• Negative Controls:

  • Isotype control: Use FITC-conjugated rabbit IgG at the same concentration as your RBFOX2 antibody to assess non-specific binding .

  • No primary antibody control: Apply only buffer without primary antibody to evaluate background from secondary reagents or tissue autofluorescence.

  • RBFOX2 knockout or knockdown samples: If available, use tissue or cells with confirmed RBFOX2 depletion to confirm specificity.

• Positive Controls:

  • Known RBFOX2-positive tissues: Include retinal sections (ganglion cells, horizontal cells), developing heart tissue, or other tissues with confirmed RBFOX2 expression .

  • Overexpression systems: Cells transfected with RBFOX2 expression constructs can serve as strong positive controls.

• Specificity Validation:

  • Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (residues 8-110 of human RBFOX2) to confirm signal specificity .

  • Western blot validation: Confirm that the antibody recognizes the expected ~50-55 kDa RBFOX2 protein band.

  • Cross-reactivity assessment: Test the antibody on tissues from multiple species if working with non-human models (confirmed reactivity with human, potential cross-reactivity with mouse based on sequence homology) .

• Technical Controls:

  • Titration series: Test multiple antibody dilutions (1:50, 1:100, 1:200, 1:500) to determine optimal signal-to-noise ratio.

  • Incubation time optimization: Compare different incubation periods to find the optimal balance between specific signal and background.

Document all validation steps meticulously to establish confidence in your experimental results and include representative images of controls in your research publications.

How can I quantify RBFOX2 expression levels using the FITC-conjugated antibody?

To accurately quantify RBFOX2 expression levels using FITC-conjugated antibody, employ these methodological approaches:

• Flow Cytometry Quantification:

  • Prepare single-cell suspensions from tissues of interest using gentle enzymatic digestion.

  • Fix cells with 2-4% paraformaldehyde and permeabilize with 0.1-0.3% Triton X-100 or saponin.

  • Stain with optimized concentration of RBFOX2 Antibody, FITC conjugated.

  • Analyze using flow cytometry with appropriate FITC detection settings.

  • Quantify mean fluorescence intensity (MFI) and percentage of RBFOX2-positive cells.

  • Include calibration beads with known fluorophore quantities to convert arbitrary units to molecules of equivalent soluble fluorochrome (MESF).

• Immunofluorescence Image Analysis:

  • Capture multiple representative images using consistent exposure settings.

  • Use image analysis software (ImageJ/FIJI, CellProfiler) to:

    • Define nuclear regions as regions of interest (ROIs) based on DAPI staining.

    • Measure FITC intensity within nuclear ROIs (where RBFOX2 is predominantly localized).

    • Subtract background fluorescence from regions without cells.

    • Calculate average intensity per nucleus and distribution of intensities across cell populations.

• Quantitative Considerations:

  • Establish a standard curve using samples with known RBFOX2 expression levels.

  • Account for tissue autofluorescence by including unstained controls.

  • Normalize RBFOX2 signal to housekeeping proteins when comparing across samples.

  • For developmental studies, track nuclear RBFOX2 intensity changes across timepoints (E12 through postnatal stages) .

• Relative vs. Absolute Quantification:

  • For relative quantification, compare RBFOX2 expression between experimental groups using consistent acquisition parameters.

  • For absolute quantification, implement quantitative fluorescence calibration using standards of known fluorophore concentrations.

This multimodal approach provides robust quantification of RBFOX2 expression patterns and enables reliable comparison across experimental conditions.

How can I investigate RBFOX2's role in alternative splicing using the FITC-conjugated antibody?

To investigate RBFOX2's role in alternative splicing using RBFOX2 Antibody, FITC conjugated, implement this comprehensive research strategy:

• Cross-linking Immunoprecipitation coupled with RNA-sequencing (CLIP-seq):

  • Use RBFOX2 Antibody for immunoprecipitation of RBFOX2-RNA complexes after UV cross-linking.

  • While the FITC conjugate isn't typically used for CLIP-seq, paralleling experiments with the unconjugated version of the same antibody clone will allow correlation with imaging data.

  • Sequence precipitated RNA to identify direct RBFOX2 binding targets containing the 5'-UGCAUGU-3' motif .

  • Analyze binding sites relative to alternatively spliced exons to predict regulatory outcomes (downstream binding promotes exon inclusion; upstream binding promotes exclusion).

• Combinatorial Imaging and Transcriptomics:

  • Perform immunofluorescence with FITC-conjugated RBFOX2 antibody to identify cells with high nuclear RBFOX2 expression.

  • Use laser capture microdissection to isolate RBFOX2-high versus RBFOX2-low cells.

  • Conduct RNA-seq on isolated populations to identify differentially spliced transcripts.

  • Focus analysis on alternative exons with adjacent UGCAUGU motifs.

• Experimental Validation of RBFOX2 Splicing Targets:

  • Design minigene splicing reporters containing candidate alternative exons and flanking introns with RBFOX2 binding sites.

  • Transfect reporters into cells with modulated RBFOX2 levels (overexpression or knockdown).

  • Analyze splicing outcomes using RT-PCR with primers flanking the alternative exon.

  • Correlate RBFOX2 nuclear levels (measured by FITC-conjugated antibody) with splicing efficiency.

• Analysis of Cell-Specific Splicing Networks:

  • Combine RBFOX2 immunofluorescence with cell-type-specific markers (e.g., for retinal ganglion cells or cardiac cells).

  • Correlate RBFOX2 expression levels with specific splicing patterns in different cell populations.

  • Investigate whether RBFOX2-dependent splicing networks differ between tissues like retina and heart .

This multifaceted approach leverages the FITC-conjugated antibody for cellular and subcellular localization studies while integrating molecular techniques to comprehensively map RBFOX2-dependent splicing regulatory networks.

What methodologies can be employed to study the role of RBFOX2 in heart development and disorders?

To investigate RBFOX2's role in heart development and disorders, implement these advanced methodological approaches:

• Conditional Knockout Models and Phenotypic Analysis:

  • Generate cardiac-specific RBFOX2 conditional knockout models using Cre-lox technology with cardiac-specific promoters like Nkx2.5-Cre .

  • Perform comprehensive cardiovascular phenotyping:

    • Non-invasive imaging (echocardiography, micro-CT) to assess cardiac chamber formation and function.

    • Histological analysis with H&E staining and immunofluorescence using RBFOX2 Antibody, FITC conjugated, to confirm knockout and examine structural abnormalities.

    • Assess outflow tract (OFT) and chamber development at multiple embryonic stages.

  • Evaluate yolk sac vasculature formation, which shows defects in RBFOX2 mutants .

• Transcriptome and Splicing Analysis in Developing Hearts:

  • Perform RNA-sequencing on control and RBFOX2-deficient hearts at different developmental stages.

  • Analyze global alternative splicing patterns using algorithms like MISO (mixture of isoform) .

  • Focus on splicing changes in genes involved in:

    • Cytoskeletal organization

    • Cell-extracellular matrix (ECM) adhesion

    • Rho GTPase signaling/cycling

  • Validate key splicing events using RT-PCR and correlate with RBFOX2 expression levels as visualized by immunofluorescence.

• Cellular Process Investigation:

  • Evaluate cell cycle progression using phospho-Histone H3 immunostaining and flow cytometry .

  • Assess endocardial-mesenchymal transition (Endo-MT) efficiency, a process dependent on cell-ECM adhesion.

  • Analyze Rho GTPase activity using FRET-based biosensors in control and RBFOX2-deficient cardiac cells.

  • Examine cell-ECM adhesion by immunostaining for focal adhesion proteins like vinculin .

• Translational Applications:

  • Screen for RBFOX2 mutations in patients with congenital heart defects, particularly hypoplastic left heart syndrome (HLHS) .

  • Develop antisense oligonucleotides to modulate RBFOX2-dependent alternative splicing events in Rho GTPase cycling genes.

  • Generate induced pluripotent stem cells (iPSCs) from HLHS patients with RBFOX2 mutations and differentiate them into cardiomyocytes to study molecular pathology.

This integrated approach enables comprehensive investigation of RBFOX2's molecular mechanisms in cardiac development and provides potential therapeutic targets for congenital heart disorders.

How can I design experiments to investigate interactions between RBFOX2 and other RNA binding proteins?

To investigate interactions between RBFOX2 and other RNA binding proteins, implement these sophisticated experimental approaches:

• Co-immunoprecipitation (Co-IP) and Proximity Ligation Assays (PLA):

  • Use RBFOX2 Antibody (unconjugated version of the same clone as the FITC-conjugated antibody) for immunoprecipitation from nuclear extracts.

  • Probe Western blots for potential interacting partners like ER-alpha, RBPMS, and MBNL1/2 .

  • Confirm interactions in situ using Proximity Ligation Assay combining RBFOX2 Antibody, FITC conjugated with antibodies against candidate interactors.

  • Quantify PLA signals in different cell types, especially in tissues where RBFOX2 is highly expressed (retinal neurons, cardiac tissues) .

• RNA-Dependent vs. RNA-Independent Interactions:

  • Perform Co-IP experiments with and without RNase treatment to distinguish direct protein-protein interactions from RNA-mediated associations.

  • For RNA-dependent interactions, identify bridging RNA species using RNA immunoprecipitation followed by sequencing (RIP-seq).

  • Map interaction domains through deletion mutant analysis of RBFOX2 and partner proteins.

• Chromatin Association Studies:

  • Investigate RBFOX2's role in transcriptional regulation via chromatin-associated nascent RNA by combining RBFOX2 immunoprecipitation with chromatin isolation techniques .

  • Examine functional interplay between RBFOX2 and polycomb repressive complex 2 (PRC2) through co-immunoprecipitation and ChIP-seq for H3K27me3 marks in control and RBFOX2-depleted cells .

  • Use FITC-conjugated RBFOX2 antibody for immunofluorescence co-localization studies with PRC2 components.

• Competitive Binding Analysis:

  • Investigate competition between RBFOX2 and U2AF2 for binding to 3'-splice sites using:

    • In vitro binding assays with purified proteins and labeled RNA substrates.

    • RNA-EMSA (Electrophoretic Mobility Shift Assay) with increasing concentrations of competitors.

    • FRET-based approaches to measure binding kinetics and displacement.

  • Correlate binding competition with alternative splicing outcomes measured by minigene assays.

• Tissue-Specific Interaction Networks:

  • Compare RBFOX2 interaction partners between neural tissues (retina) and cardiac tissues .

  • Investigate developmental changes in interaction patterns by analyzing samples from different embryonic stages.

  • Connect interaction differences to tissue-specific alternative splicing programs.

This multifaceted approach will provide comprehensive insights into RBFOX2's interactions with other RNA-binding proteins and how these interactions contribute to tissue-specific RNA regulatory networks during development.

What experimental approaches can reveal RBFOX2's role in retinal development and visual function?

To comprehensively investigate RBFOX2's role in retinal development and visual function, implement these advanced experimental approaches:

• Developmental Expression Profiling:

  • Use RBFOX2 Antibody, FITC conjugated, for immunofluorescence analysis across developmental timepoints from E12 through adulthood .

  • Co-stain with markers for retinal progenitor cells and differentiating retinal neurons to track RBFOX2 expression during cell fate specification.

  • Perform quantitative image analysis to measure changes in nuclear RBFOX2 levels during development.

  • Compare RBFOX2 and RBFOX1 expression patterns, noting their different subcellular localization dynamics (RBFOX2 remains nuclear throughout development, while RBFOX1 transitions from cytoplasmic to nuclear at around P0) .

• Cell Type-Specific RBFOX2 Knockdown:

  • Design shRNA or CRISPR/Cas9 approaches targeting RBFOX2 with retina-specific promoters.

  • Use in vivo electroporation to deliver knockdown constructs to developing retina.

  • Analyze effects on:

    • Cell fate specification of retinal ganglion cells, horizontal cells, and amacrine cells.

    • Morphology and dendritic arborization of RBFOX2-expressing neurons.

    • Synaptic organization in the inner and outer plexiform layers.

• Visual Function Assessment:

  • Perform electroretinography (ERG) to measure retinal responses to light stimuli in control and RBFOX2-deficient retinas.

  • Conduct optokinetic response testing to assess visual function at the behavioral level.

  • Use visual-evoked potentials (VEPs) to evaluate signal transmission from retina to visual cortex.

  • Correlate functional deficits with specific cellular abnormalities in RBFOX2-deficient retinas.

• Retinal Transcriptome Analysis:

  • Perform RNA-sequencing on RBFOX2-deficient and control retinas at key developmental timepoints.

  • Analyze alternative splicing patterns using computational tools specifically designed for splicing analysis.

  • Focus on:

    • Neuron-specific splicing events

    • Genes involved in synaptogenesis

    • Ion channels and neurotransmitter receptors

  • Validate key splicing changes using RT-PCR and correlate with visual function deficits.

• Circuit-Level Analysis:

  • Use patch-clamp recording to analyze physiological properties of RBFOX2-expressing neurons.

  • Implement calcium imaging to assess activity patterns in retinal circuits with normal or reduced RBFOX2 levels.

  • Perform connectomic analysis using serial electron microscopy to evaluate synaptic organization in RBFOX2-deficient retinas.

This comprehensive approach integrates molecular, cellular, physiological, and behavioral methods to elucidate RBFOX2's multifaceted roles in retinal development and visual processing.

How can I optimize signal-to-noise ratio when using RBFOX2 Antibody, FITC conjugated in tissues with high autofluorescence?

To optimize signal-to-noise ratio when using RBFOX2 Antibody, FITC conjugated in tissues with high autofluorescence, implement these methodological approaches:

These approaches, used alone or in combination, will significantly improve signal-to-noise ratio when detecting RBFOX2 in tissues with challenging autofluorescence characteristics.

What strategies can I employ when investigating RBFOX2 in tissues where expression levels are low or variable?

When investigating RBFOX2 in tissues with low or variable expression levels, implement these methodological strategies to maximize detection sensitivity and quantification accuracy:

• Signal Amplification Techniques:

  • Though not directly applicable to FITC-conjugated antibodies, consider parallel experiments with unconjugated RBFOX2 antibody using:

    • Tyramide signal amplification (TSA) to enhance fluorescence signal by up to 100-fold.

    • Poly-HRP detection systems that provide significant signal amplification.

    • Quantum dot-conjugated secondary antibodies for improved signal stability and brightness.

  • Optimize exposure times and detector sensitivity settings during imaging to capture low-level signals without introducing excessive noise.

• Sample Enrichment Approaches:

  • Implement nuclear isolation protocols before immunostaining to concentrate RBFOX2, which is predominantly nuclear .

  • Use laser capture microdissection to isolate specific cell populations with known RBFOX2 expression.

  • Consider cell sorting (FACS) of dissociated tissue using cell type-specific surface markers to enrich for RBFOX2-expressing populations.

• Complementary Detection Methods:

  • Validate immunofluorescence findings with highly sensitive RNA detection methods:

    • RNAscope in situ hybridization for RBFOX2 mRNA detection with single-molecule sensitivity.

    • Droplet digital PCR (ddPCR) for absolute quantification of RBFOX2 transcript levels.

  • Implement Western blotting with chemiluminescent detection for protein-level validation.

  • Consider using proximity ligation assay (PLA) to detect RBFOX2 interactions with known binding partners, which can provide signal amplification for low-abundance proteins.

• Optimization for Specific Tissue Types:

  • For neural tissues: Extend fixation time to 14-16 hours at 4°C to better preserve RBFOX2 antigenicity .

  • For cardiac tissues: Implement antigen retrieval optimization specific for RBFOX2 detection in heart samples .

  • For retinal tissues: Adjust fixation protocols based on developmental stage, as RBFOX2 expression changes during retinal development .

• Quantitative Analysis Adaptations:

  • Implement background subtraction methods specific to tissues with variable autofluorescence.

  • Use internal reference standards for normalization across samples.

  • Consider ratio-based measurements (RBFOX2 to nuclear marker) rather than absolute intensity values.

  • Increase statistical power by analyzing larger sample sizes and more fields of view per sample.

These strategies collectively enhance detection sensitivity and quantification accuracy when studying RBFOX2 in challenging tissue contexts with low or variable expression patterns.

How can I distinguish between specific and non-specific signals when using RBFOX2 Antibody, FITC conjugated in my experiments?

To definitively distinguish between specific and non-specific signals when using RBFOX2 Antibody, FITC conjugated, implement these rigorous validation approaches:

• Comprehensive Control Panel Implementation:

  • Genetic Controls: Use RBFOX2 knockout/knockdown tissues where available, or siRNA-treated cells with confirmed RBFOX2 depletion.

  • Absorption Controls: Pre-incubate antibody with excess immunizing peptide (recombinant human RBFOX2 protein residues 8-110AA) to block specific binding sites.

  • Isotype Controls: Use FITC-conjugated rabbit IgG at identical concentration to assess non-specific binding from antibody constant regions.

  • Secondary-Only Controls: For multi-color staining protocols with additional primary antibodies, include wells with only secondary antibodies to assess background.

• Signal Pattern Analysis:

  • Subcellular Localization Verification: Confirm that RBFOX2 signal is predominantly nuclear, as expected based on its known localization .

  • Cell Type-Specific Expression: Verify that signal appears in expected cell types (retinal ganglion cells, horizontal cells, GABAergic amacrine cells, cardiac cells) .

  • Development-Specific Patterns: Confirm that RBFOX2 maintains nuclear localization throughout development, unlike RBFOX1 which changes localization .

  • Signal Intensity Correlation: Compare signal intensity with known expression levels in different tissues/cell types.

• Multi-Method Validation:

  • Orthogonal Detection: Validate findings using alternative detection methods like Western blotting or mass spectrometry.

  • Alternative Antibodies: Test multiple RBFOX2 antibodies targeting different epitopes to confirm signal consistency.

  • Transcript-Protein Correlation: Compare protein localization with mRNA expression using in situ hybridization techniques.

  • Cross-Species Consistency: Verify that signal patterns match known evolutionary conservation of RBFOX2 expression.

• Technical Signal Discrimination:

  • Spectral Unmixing: Use spectral imaging and linear unmixing algorithms to separate FITC signal from autofluorescence.

  • Signal Quantification: Plot signal-to-background ratios across samples and establish objective thresholds for positive staining.

  • Time-Resolved Imaging: Exploit differences in fluorescence lifetime between specific FITC signal and tissue autofluorescence.

  • Sequential Imaging: Photobleach autofluorescence before acquiring FITC channel to improve signal specificity.

• Antibody Titration and Competition Assays:

  • Perform antibody titration experiments (1:50 to 1:1000) to identify optimal concentration for specific binding.

  • Use increasing concentrations of unlabeled antibody to compete with FITC-conjugated antibody as confirmation of binding specificity.

These approaches provide a comprehensive framework for distinguishing specific RBFOX2 signal from technical artifacts and non-specific background, ensuring robust and reproducible experimental outcomes.

How can I integrate RBFOX2 Antibody staining with single-cell transcriptomics to map RNA regulatory networks?

To integrate RBFOX2 Antibody staining with single-cell transcriptomics for mapping RNA regulatory networks, implement this cutting-edge methodological workflow:

• FACS-based Integration Approach:

  • Prepare single-cell suspensions from tissues of interest (retina, heart, or other RBFOX2-expressing tissues).

  • Perform fixation and permeabilization compatible with both antibody staining and RNA preservation.

  • Stain with RBFOX2 Antibody, FITC conjugated, at optimized concentration.

  • Use FACS to isolate cells based on RBFOX2 expression levels (high, medium, low).

  • Process sorted populations for single-cell RNA sequencing using Smart-seq2 or other full-length protocols that can detect splicing isoforms.

  • Analyze alternative splicing patterns correlated with RBFOX2 expression levels, focusing on exons with adjacent UGCAUGU motifs .

• CITE-seq/REAP-seq Adaptation:

  • Modify CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) protocols to include RBFOX2 antibody.

  • Tag RBFOX2 antibody with oligonucleotide barcodes instead of FITC for sequencing-based quantification.

  • Simultaneously capture surface protein expression, RBFOX2 levels, and transcriptome/splicing information from individual cells.

  • Implement computational pipelines to correlate RBFOX2 protein levels with alternative splicing events.

• Spatial Transcriptomics Integration:

  • Perform RBFOX2 immunofluorescence on tissue sections destined for spatial transcriptomics.

  • Capture high-resolution images of RBFOX2 FITC signal before proceeding with spatial transcriptomics protocols.

  • Register RBFOX2 protein expression data with spatially resolved transcriptome data.

  • Analyze spatial co-variation between RBFOX2 protein levels and alternative splicing patterns of target transcripts.

  • Map tissue-specific RNA regulatory networks in anatomical context, particularly in the developing heart and retina .

• Advanced Computational Analysis:

  • Implement machine learning approaches to identify RNA features that predict RBFOX2-dependent regulation.

  • Construct gene regulatory networks incorporating:

    • RBFOX2 binding motifs

    • Alternative splicing events

    • Expression levels of splicing targets

    • Cell type-specific splicing programs

  • Develop predictive models for RBFOX2's impact on tissue-specific development based on integrated protein-RNA data.

• Validation and Perturbation Studies:

  • Select key predicted RBFOX2 targets for validation using minigene splicing reporters.

  • Implement CRISPR-based manipulation of RBFOX2 binding sites in endogenous targets.

  • Correlate alternative splicing changes with developmental or functional phenotypes, particularly in heart development and retinal function .

This integrated approach combines protein localization data with transcriptome-wide splicing information at single-cell resolution, enabling unprecedented insights into RBFOX2-dependent RNA regulatory networks across different tissues and developmental stages.

What emerging technologies can be combined with RBFOX2 Antibody, FITC conjugated to study dynamic RNA-protein interactions in live cells?

To study dynamic RNA-protein interactions involving RBFOX2 in live cells, combine RBFOX2 Antibody, FITC conjugated with these emerging technologies:

• Intracellular Antibody Delivery Systems:

  • While conventional antibodies including FITC-conjugated RBFOX2 antibody cannot penetrate live cells, implement:

    • Cell-penetrating peptide conjugation to RBFOX2 antibody for intracellular delivery.

    • Electroporation-based delivery of RBFOX2 antibody into live cells.

    • Lipid-based transfection of RBFOX2 antibody fragments (Fab fragments) for live imaging.

  • Alternatively, use genetically encoded intrabodies derived from RBFOX2 antibody sequences, fused to fluorescent proteins for live-cell visualization.

• RNA Visualization Technologies:

  • Implement MS2/MS2 coat protein or similar systems to tag RBFOX2 target RNAs.

  • Use CRISPR-Cas13 RNA tracking systems with different fluorophores to visualize target RNAs.

  • Apply Suntag or similar amplification systems to enhance RNA signal detection.

  • Combine with RBFOX2-specific fluorescent labeling to visualize co-localization and interaction dynamics.

• Advanced Microscopy Techniques:

  • Apply FRET (Förster Resonance Energy Transfer) between FITC-labeled RBFOX2 antibody and RNA-binding dyes to measure direct interactions.

  • Implement FLIM (Fluorescence Lifetime Imaging Microscopy) to detect RBFOX2-RNA binding independent of concentration effects.

  • Use spt-PALM (single-particle tracking photoactivated localization microscopy) to track individual RBFOX2 molecules in the nucleus.

  • Apply lattice light-sheet microscopy for high-speed 3D imaging of RBFOX2 dynamics with minimal phototoxicity.

• Optogenetic and Chemogenetic Systems:

  • Develop optogenetic RBFOX2 variants that can be activated or inactivated with light to study temporal dynamics of splicing regulation.

  • Create split-protein complementation systems where RBFOX2 activity is controlled by small molecules.

  • Combine with RNA aptamer systems that fluoresce upon binding specific small molecules to visualize RBFOX2-RNA interactions.

• Microfluidic and Biophysical Approaches:

  • Design microfluidic chambers for real-time observation of RBFOX2-RNA interactions with controlled delivery of transcription and splicing factors.

  • Apply acoustic force spectroscopy or optical tweezers to measure binding forces between RBFOX2 and target RNAs.

  • Implement liquid-liquid phase separation (LLPS) assays to study how RBFOX2 contributes to formation of splicing regulatory condensates.

While direct application of FITC-conjugated antibodies in live cells presents technical challenges, these advanced technologies provide innovative approaches to study RBFOX2 dynamics and RNA interactions in living systems, offering insights into the temporal regulation of alternative splicing during development and disease processes.

How can computational modeling be integrated with RBFOX2 Antibody staining data to predict splicing outcomes in developmental disorders?

To integrate computational modeling with RBFOX2 Antibody staining data for predicting splicing outcomes in developmental disorders, implement this innovative methodological framework:

• Quantitative Image Analysis Pipeline:

  • Develop automated image analysis workflows to extract nuclear RBFOX2 intensities from FITC-conjugated antibody staining across tissue sections.

  • Implement cell segmentation algorithms to identify individual nuclei and quantify RBFOX2 levels on a cell-by-cell basis.

  • Create spatial maps of RBFOX2 expression across developmental timepoints and tissue regions.

  • Correlate RBFOX2 expression patterns with developmental markers and tissue morphology.

• Integrative Modeling of Splicing Regulation:

  • Construct mathematical models incorporating:

    • RBFOX2 concentration (from immunostaining data)

    • RNA binding affinities (from CLIP-seq or similar data)

    • Position-dependent effects of RBFOX2 binding relative to alternative exons

    • Competition with other splicing factors

    • Tissue-specific cofactors

  • Develop deep learning approaches trained on existing RBFOX2-dependent splicing data from heart and retinal tissues .

  • Create Bayesian networks to predict exon inclusion/exclusion probabilities based on RBFOX2 levels and binding site characteristics.

• Validation and Refinement Cycle:

  • Test model predictions using minigene splicing reporters with systematic mutations in RBFOX2 binding sites.

  • Validate in vivo using tissues with gradient RBFOX2 expression or partial knockdown models.

  • Refine models based on experimental validation, incorporating RNA structural features and cooperative binding effects.

  • Implement active learning approaches to identify the most informative experiments for model improvement.

• Disease-Specific Applications:

  • Apply models to predict splicing changes in hypoplastic left heart syndrome (HLHS) based on RBFOX2 mutations or expression changes .

  • Extend to other developmental disorders with RBFOX2 dysfunction, including neurological conditions associated with retinal abnormalities .

  • Incorporate patient-specific RBFOX2 mutations to create personalized splicing outcome predictions.

  • Design computational screens for small molecules or antisense oligonucleotides that could correct aberrant splicing in disease contexts.

• Multi-scale Integration:

  • Develop multi-scale models connecting molecular events (RBFOX2-regulated splicing) to cellular phenotypes (cell adhesion, migration) and tissue-level outcomes (heart chamber formation, retinal circuitry).

  • Implement agent-based modeling approaches where individual cells with varying RBFOX2 levels interact within developing tissues.

  • Create predictive frameworks for developmental trajectories based on early RBFOX2 expression patterns and splicing signatures.