IRF3 Antibody, FITC conjugated

What is IRF3 and why is it important in immune research?

IRF3 (Interferon Regulatory Factor 3) is a key transcriptional regulator of type I interferon (IFN)-dependent immune responses that plays a critical role in the innate immune response against DNA and RNA viruses. It regulates the transcription of type I IFN genes (IFN-alpha and IFN-beta) and IFN-stimulated genes (ISG) by binding to an interferon-stimulated response element (ISRE) in their promoters. IRF3 acts as a more potent activator of the IFN-beta (IFNB) gene than the IFN-alpha (IFNA) gene and plays a critical role in both early and late phases of IFNA/B gene induction . In uninfected cells, IRF3 exists in an inactive form in the cytoplasm, but following viral infection, double-stranded RNA (dsRNA), or toll-like receptor (TLR) signaling, it undergoes phosphorylation by kinases such as IKBKE and TBK1, inducing conformational changes that enable dimerization and nuclear translocation .

What are the key specifications of commercially available IRF3-FITC antibodies?

Commercial IRF3-FITC antibodies exhibit varying specifications based on manufacturer and target applications. Most are polyclonal rabbit IgG antibodies with fluorescein isothiocyanate (FITC) conjugation, providing excitation at approximately 495-499 nm and emission at 515-519 nm . These antibodies are typically optimized for laser line 488 nm detection systems . Species reactivity ranges from human-specific to multi-species (human, mouse, rat, monkey) antibodies, with some having predicted reactivity to additional species . The immunogens used vary by manufacturer, with some targeting the carboxy terminus of human IRF3 , while others target specific phosphorylation sites like Ser396 or use recombinant proteins encompassing larger regions (e.g., amino acids 1-419) .

What applications are IRF3-FITC antibodies validated for?

IRF3-FITC antibodies have been validated for multiple research applications, including:

• Western Blot (WB)

• Enzyme-Linked Immunosorbent Assay (ELISA)

• Immunocytochemistry/Immunofluorescence (ICC/IF)

• Immunohistochemistry with paraffin-embedded samples (IHC-P)

• Immunohistochemistry with frozen samples (IHC-F)

• Flow cytometry

Optimal dilutions for these applications should be experimentally determined for each specific antibody and experimental system . For most applications, these antibodies can detect both inactive cytoplasmic IRF3 and the activated, phosphorylated forms that translocate to the nucleus during immune responses .

How can IRF3-FITC antibodies be used to monitor temporal dynamics of IRF3 activation?

IRF3-FITC antibodies can effectively monitor the temporal dynamics of IRF3 activation through time-course imaging experiments. Following stimulation with viral agonists like Sendai Virus (SenV), IRF3 undergoes characteristic activation, turnover, and recovery patterns that can be visualized over 48-hour periods . For optimal temporal monitoring, researchers should:

• Establish appropriate baseline measurements with resting cells

• Implement time-point sampling at 0, 8, 16, 24, and 48 hours post-stimulation

• Utilize confocal microscopy to monitor IRF3-FITC subcellular localization shifts

• Complement imaging with flow cytometry for quantitative cellular analysis

• Consider dual staining with nuclear markers to quantify nuclear translocation percentages

This approach reveals the characteristic "laddering" pattern of IRF3 phosphorylation species and the depletion/recovery cycle of resting IRF3 following immune stimulation . For quantitative analysis, the Amnis Imagestream instrument enables high-throughput assessment of IRF3 protein levels and activation state on a per-cell basis .

What are the key considerations when comparing phospho-specific versus total IRF3-FITC antibodies?

The selection between phospho-specific and total IRF3-FITC antibodies significantly impacts experimental design and data interpretation:

For phospho-specific antibodies like IRF3 (Ser396), the immunogen typically consists of a KLH-conjugated synthetic phosphopeptide derived from human IRF3 around the phosphorylation site . These antibodies provide more mechanistic insight into IRF3 activation but require careful sample handling to preserve phosphorylation status. In contrast, total IRF3 antibodies may target the carboxy terminus or use recombinant full-length proteins as immunogens, capturing all forms of the protein regardless of modification state.

How can IRF3-FITC antibodies be integrated into multiparameter flow cytometry panels?

Integrating IRF3-FITC antibodies into multiparameter flow cytometry requires strategic panel design that considers both spectral compatibility and biological relevance:

• Spectral considerations: FITC emission (515-519 nm) overlaps with PE, requiring appropriate compensation controls. Optimal pairing involves fluorophores with minimal spectral overlap like APC (660 nm) and BV421 (421 nm).

• Antibody titration: Each IRF3-FITC antibody must be titrated specifically for flow cytometry to determine optimal signal-to-noise ratio. Typically start with manufacturer-recommended concentrations and test serial dilutions.

• Fixation/permeabilization: Optimize protocols that preserve FITC signal while enabling intracellular access. Methanol-based protocols can reduce FITC signal, while paraformaldehyde with saponin or Triton X-100 often provides better results.

• Biologically relevant markers: Pair IRF3-FITC with surface markers for cell identification (CD3, CD4, CD8, CD19) and additional markers in the IRF3 pathway:

  • TBK1/IKKε (upstream kinases)

  • Type I interferon receptors

  • STAT1/2 (downstream effectors)

  • Viral sensors (RIG-I, MDA5, cGAS)

• Controls: Include biological controls (unstimulated vs. stimulated with SenV or poly I:C) alongside fluorescence minus one (FMO) controls.

When properly designed, these panels can simultaneously assess IRF3 activation status across multiple immune cell populations in response to viral stimulation or other immune challenges.

What are the optimal fixation and permeabilization protocols for IRF3-FITC immunofluorescence?

Optimal fixation and permeabilization protocols for IRF3-FITC immunofluorescence vary based on the specific antibody and experimental goals:

• For detection of total IRF3 distribution:

  • Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • Permeabilize with 0.1-0.5% Triton X-100 for 5-10 minutes

  • Block with 5% normal serum (matching secondary antibody host) for 30-60 minutes

  • Incubate with IRF3-FITC at optimal dilution (typically 1:50 to 1:200) for 1-2 hours or overnight at 4°C

  • Counterstain nucleus with DAPI and mount with anti-fade mounting medium

• For phospho-specific IRF3 detection:

  • Include phosphatase inhibitors in all buffers

  • Consider methanol fixation (-20°C for 10 minutes) for better epitope accessibility

  • Alternatively, use 4% paraformaldehyde followed by methanol post-fixation

  • Extend blocking time to 2 hours to reduce background

  • Include bovine serum albumin (1%) in antibody diluent

• For nuclear translocation studies:

  • Process stimulated and unstimulated samples identically

  • Consider time-course experiments with defined time points

  • Use confocal microscopy for precise subcellular localization

  • Quantify nuclear/cytoplasmic ratio across multiple cells

All protocols should avoid repeated freeze/thaw cycles of the antibody and minimize exposure to light to preserve FITC signal integrity . Storage at either 4°C (short-term) or -20°C (long-term, aliquoted) is recommended to maintain antibody performance .

How should researchers validate specificity of IRF3-FITC antibodies for their experimental system?

Rigorous validation of IRF3-FITC antibodies is essential for ensuring reliable research outcomes. A comprehensive validation approach should include:

• Positive controls:

  • Cell lines with known IRF3 expression (e.g., HeLa cells)

  • Stimulation with established IRF3 activators (SenV, poly I:C, LPS)

  • Recombinant IRF3 protein as Western blot standard

• Negative controls:

  • IRF3 knockout/knockdown cell lines

  • Isotype-matched FITC-conjugated control antibodies

  • Pre-absorption with immunizing peptide to confirm specificity

• Cross-validation techniques:

  • Parallel testing with multiple anti-IRF3 antibodies targeting different epitopes

  • Comparison of results using different detection methods (Western blot, immunofluorescence, flow cytometry)

  • Correlation of protein detection with mRNA expression levels

• Species-specific validation:

  • Confirm reactivity in your species of interest, especially if working with models other than human or mouse

  • Note that sequence homology between species can vary (e.g., 86% bovine homology to human IRF3)

  • Consider species-specific positive controls

• Isoform considerations:

  • At least three IRF3 isoforms are known to exist

  • Determine which isoforms your antibody detects

  • Confirm antibody specificity using recombinant isoforms if possible

Validation experiments should be documented and included in publications to support the reliability of findings. Additionally, researchers should be aware that different lots of the same antibody may show slight variation in performance.

What protocols should be followed for tracking IRF3 nuclear translocation using FITC-conjugated antibodies?

For effectively tracking IRF3 nuclear translocation using FITC-conjugated antibodies, researchers should follow these methodological steps:

• Cell preparation:

  • Culture cells on coverslips or in chamber slides

  • Include appropriate stimulation conditions (e.g., 200 HAU/mL SenV for 0-48 hours)

  • Prepare parallel unstimulated controls

• Fixation and immunostaining:

  • Fix cells with 4% paraformaldehyde at specified time points

  • Permeabilize with 0.1% Triton X-100

  • Block with 5% normal serum

  • Stain with IRF3-FITC antibody (optimal dilution determined experimentally)

  • Counterstain nucleus with DAPI or Hoechst

• Confocal microscopy acquisition:

  • Capture z-stack images to ensure complete nuclear volume assessment

  • Use identical acquisition parameters across all samples

  • Include both FITC and nuclear channels

  • Image at least 10-15 fields per condition with >50 cells total

• Quantitative analysis:

  • Measure nuclear/cytoplasmic FITC intensity ratio

  • Score cells as positive for nuclear translocation when nuclear signal exceeds cytoplasmic

  • Calculate percentage of cells with nuclear IRF3 for each condition

  • Plot temporal dynamics showing translocation kinetics

• Alternative flow cytometry approach:

  • Fix and permeabilize cells at desired time points

  • Stain with IRF3-FITC and nuclear dye

  • Analyze using imaging flow cytometry (e.g., Amnis Imagestream)

  • Quantify co-localization of IRF3-FITC with nuclear signal

  • Process high-throughput data on a per-cell basis

This integrated approach enables both qualitative visualization and quantitative assessment of IRF3 translocation dynamics following stimulation, providing insights into the temporal regulation of innate immune responses .

How can researchers address weak or inconsistent IRF3-FITC antibody signals?

Weak or inconsistent IRF3-FITC antibody signals represent a common challenge that can be systematically addressed:

• Antibody optimization:

  • Titrate antibody concentration over a wide range (1:10 to 1:5000)

  • Test extended incubation times (overnight at 4°C vs. 1-2 hours at room temperature)

  • Consider signal amplification techniques (tyramide signal amplification)

  • Ensure antibody has not degraded (avoid repeated freeze/thaw cycles, protect from light)

• Sample preparation improvements:

  • Optimize fixation duration (10-20 minutes with 4% paraformaldehyde)

  • Test multiple permeabilization reagents (Triton X-100, saponin, methanol)

  • Include protease and phosphatase inhibitors in all buffers

  • Reduce background with extended blocking (2 hours with 5-10% serum)

  • Consider antigen retrieval for tissue sections (citrate buffer, pH 6.0)

• Technical considerations:

  • Verify microscope/cytometer settings (laser alignment, filter sets)

  • Adjust detector gain and sensitivity specifically for FITC

  • Minimize photobleaching during acquisition

  • Keep all processing steps identical between experiments

• Biological variables:

  • Confirm IRF3 expression levels in your cell type/tissue

  • Include positive controls with known IRF3 induction (SenV infection)

  • Consider cell cycle effects on IRF3 expression

  • Account for activation state kinetics (IRF3 levels change post-stimulation)

If signals remain problematic after optimization, consider switching to a different IRF3 antibody clone or manufacturer, or explore alternative detection methods like enzymatic amplification systems.

How should researchers interpret differences in IRF3 activation patterns across cell types?

Interpreting differences in IRF3 activation patterns across cell types requires careful consideration of both biological and technical factors:

• Biological interpretation framework:

  • Cell-type specific baseline IRF3 expression levels vary significantly

  • IRF3 activation thresholds differ between immune and non-immune cells

  • Temporal dynamics of activation/deactivation follow cell-type specific patterns

  • Upstream sensor expression (RIG-I, MDA5, cGAS, TLRs) varies by cell type

  • Negative regulators of IRF3 signaling show tissue-specific expression

• Quantitative comparison approaches:

  • Normalize IRF3 activation to total IRF3 levels per cell type

  • Compare fold-change in nuclear translocation rather than absolute percentages

  • Analyze activation kinetics (time to peak, duration of activation)

  • Assess downstream gene induction relative to IRF3 activation

• Technical considerations for cross-cell type comparisons:

  • Use identical stimulation conditions and antibody concentrations

  • Account for cell-type differences in antibody penetration

  • Consider autofluorescence differences between cell types

  • Verify specificity in each cell type independently

• Integrative analysis:

  • Correlate IRF3 activation with functional outcomes (IFN production)

  • Connect differences to known biological functions of each cell type

  • Consider evolutionary adaptations in different tissues

When properly controlled and normalized, cross-cell type comparisons of IRF3 activation patterns can reveal fundamental insights into tissue-specific innate immune responses and their regulation. These differences often reflect evolutionary adaptations to specific pathogen pressures in different tissues.

What are the critical controls for distinguishing specific from non-specific IRF3-FITC antibody binding?

Robust experimental design for IRF3-FITC antibody applications requires comprehensive controls to distinguish specific from non-specific binding:

• Essential negative controls:

  • Isotype-matched FITC-conjugated control antibody at identical concentration

  • Secondary antibody-only control (if using indirect detection)

  • IRF3 knockout or knockdown samples when available

  • Blocking peptide competition (pre-incubation with immunizing peptide)

• Positive controls:

  • Cell lines with confirmed IRF3 expression (e.g., HeLa)

  • Stimulated samples showing expected IRF3 activation pattern

  • Recombinant IRF3 protein (for Western blot validation)

• Technical controls:

  • Autofluorescence control (unstained samples)

  • Fixation control (impact of fixation method on background)

  • Concentration gradient testing to identify optimal signal-to-noise ratio

• Validation approaches:

  • Cross-validation with non-conjugated IRF3 antibodies

  • Orthogonal methods (Western blot, mass spectrometry)

  • Correlation with mRNA expression data

  • Expected subcellular localization patterns before/after stimulation

• Quantitative assessment:

  • Signal-to-noise ratio calculation

  • Coefficient of variation across technical replicates

  • Consistency across independent experiments

  • Alignment with expected molecular weight and characteristics

Researchers should systematically document these controls and include appropriate control data in publications to establish the specificity and reliability of their IRF3-FITC antibody applications.

How can IRF3-FITC antibodies be used for high-throughput screening applications?

IRF3-FITC antibodies offer significant potential for high-throughput screening (HTS) applications in immunology and virology research:

• Assay development for HTS platforms:

  • Optimize IRF3-FITC staining for microplate formats (96/384-well)

  • Establish automated imaging workflows using high-content analyzers

  • Develop quantitative readouts (nuclear/cytoplasmic ratio, percent activation)

  • Implement machine learning algorithms for automated classification

  • Validate with known IRF3 activators and inhibitors

• Screening applications:

  • Anti-viral drug discovery targeting IRF3-dependent pathways

  • Identification of novel pattern recognition receptor agonists

  • Characterization of pathogen immune evasion mechanisms

  • Evaluation of adjuvant candidates for vaccine development

  • Discovery of immunomodulatory compounds

• Advanced flow cytometry approaches:

  • Flow cytometry-based screening using the Amnis Imagestream for high-throughput, high-content analysis

  • Multi-parameter analysis of IRF3 activation alongside other immune markers

  • Single-cell isolation of IRF3-activated populations for downstream analysis

• Assay performance metrics:

  • Z-factor >0.5 for robust screening assays

  • Signal window >2-fold between positive and negative controls

  • Coefficient of variation <20% for technical replicates

  • DMSO tolerance assessment for compound screening

• Data analysis frameworks:

  • Multivariate analysis of activation patterns

  • Dose-response profiling for hit characterization

  • Temporal dynamics analysis for mechanistic insights

  • Integration with other pathway readouts for network-level understanding

The development of IRF3-FITC-based high-throughput assays enables systematic exploration of innate immune regulation and provides platforms for discovering novel therapeutic approaches targeting viral infections and inflammatory disorders.

What methodological approaches enable simultaneous detection of IRF3 dimerization and nuclear translocation?

Advanced methodological approaches that enable simultaneous assessment of IRF3 dimerization and nuclear translocation provide deeper mechanistic insights:

• Imaging-based approaches:

  • Proximity ligation assay (PLA) combined with IRF3-FITC immunostaining

  • Förster resonance energy transfer (FRET) using IRF3-FITC paired with IRF3-Texas Red

  • Bimolecular fluorescence complementation with split fluorescent proteins fused to IRF3

  • Super-resolution microscopy to visualize IRF3 oligomeric structures

• Biochemical approaches with imaging correlation:

  • Native PAGE followed by Western blot to detect IRF3 dimers

  • Cross-linking coupled with immunoprecipitation

  • Size exclusion chromatography of cell lysates

  • Correlative microscopy linking biochemical analysis with imaging

• Live-cell tracking systems:

  • Dual-color live-cell imaging with differentially tagged IRF3 constructs

  • Photoactivatable or photoconvertible IRF3 fusion proteins

  • Light-sheet microscopy for 3D visualization of dynamics

  • Long-term tracking with minimal phototoxicity

• Quantitative analysis frameworks:

  • Correlation of dimerization intensity with nuclear accumulation

  • Temporal sequence analysis (dimerization preceding translocation)

  • Single-cell trajectory analysis linking both phenomena

  • Mathematical modeling of the relationship between dimerization and translocation

• Integrative multi-omics approaches:

  • Combine imaging with phosphoproteomics

  • Link to chromatin immunoprecipitation data on IRF3 binding

  • Correlate with transcriptional readouts of IRF3 activity

  • Single-cell multi-modal analysis combining protein states with transcriptional outputs

These methodological approaches reveal the mechanistic coupling between IRF3 dimerization (a key activation step) and its subsequent nuclear translocation, providing insights into the regulation of this critical innate immune pathway.

How can researchers optimize IRF3-FITC antibodies for intravital imaging applications?

Intravital imaging using IRF3-FITC antibodies presents unique challenges requiring specialized optimization strategies:

• Antibody format optimization:

  • Consider Fab or nanobody alternatives for better tissue penetration

  • Test different FITC conjugation ratios for optimal signal-to-background

  • Evaluate stability under physiological temperature and pH

  • Validate antibody specificity in whole-animal contexts

  • Optimize dosing for sufficient target detection with minimal background

• Administration approaches:

  • Intravenous delivery with optimal circulation time assessment

  • Direct administration to tissues of interest

  • Loading of ex vivo cells followed by adoptive transfer

  • Microinjection techniques for localized delivery

• Imaging window considerations:

  • Surgical window creation with minimal tissue disruption

  • Use of dorsal skin fold chambers for longitudinal studies

  • Transparent zebrafish or xenograft models

  • Organ-specific imaging windows for specialized applications

• Microscopy adaptations:

  • Two-photon microscopy to minimize phototoxicity and increase penetration

  • Resonant scanning for rapid acquisition

  • Adaptive optics to correct for tissue-induced aberrations

  • Light-sheet approaches for reduced photobleaching

• Signal optimization strategies:

  • Autofluorescence elimination through spectral unmixing

  • Signal amplification techniques compatible with living systems

  • Respiratory and cardiac gating for motion artifact reduction

  • Image registration and stabilization algorithms

• Analysis frameworks:

  • 4D tracking of IRF3 dynamics in vivo

  • Cell-type specific quantification using additional markers

  • Correlation with pathogen localization or tissue damage

  • Integration with physiological parameters

These strategies enable the translation of in vitro IRF3-FITC antibody applications to powerful in vivo imaging approaches, opening new avenues for studying innate immune regulation in physiologically relevant contexts.