STAT4 Antibody, FITC conjugated

What is STAT4 and why is it an important research target?

STAT4 is a transcription factor belonging to the Signal Transducer and Activator of Transcription (STAT) family, sharing 52% similarity with STAT1 and 47% with STAT3 . It functions as a critical regulator in cellular growth, differentiation, and immune responses, with expression primarily restricted to the thymus, spleen, and testis . STAT4 plays a pivotal role in the differentiation of T-helper 1 cells and interferon-gamma (IFN-γ) production, making it essential for effective host defense against intracellular pathogens . Additionally, STAT4 participates in multiple neutrophil functions, including chemotaxis and production of neutrophil extracellular traps . Its central role in immune regulation makes STAT4 a valuable target for studying inflammatory and autoimmune conditions as well as immune responses to infections.

How do FITC-conjugated STAT4 antibodies differ from other conjugates in research applications?

FITC-conjugated STAT4 antibodies offer specific advantages for certain research applications compared to other conjugates. FITC has an excitation maximum at approximately 495 nm and emission maximum at 519 nm, producing a bright green fluorescence that is compatible with most fluorescence detection systems . When selecting between FITC and other fluorophores like phycoerythrin (PE) or various Alexa Fluor® conjugates, researchers should consider: (1) the spectral compatibility with other fluorophores in multi-color experiments, (2) the autofluorescence characteristics of the target tissue, and (3) the photostability requirements of the experimental design . FITC-conjugated antibodies are particularly valuable for immunofluorescence, flow cytometry, and immunohistochemistry applications where direct visualization of STAT4 location and quantification is needed without additional secondary antibody incubation steps .

What quality control parameters should be assessed when selecting a FITC-conjugated STAT4 antibody?

When selecting a FITC-conjugated STAT4 antibody, researchers should evaluate several key quality control parameters:

Researchers should always review validation data specific to their intended application and verify that the antibody has been tested in relevant cell types such as HepG2 cells or mouse Th1 cells as demonstrated in validation studies .

What are the optimal conditions for using FITC-conjugated STAT4 antibodies in flow cytometry?

For optimal results in flow cytometry applications using FITC-conjugated STAT4 antibodies, the following protocol parameters should be considered:

Flow cytometric analysis is particularly valuable for differentiating between Th1 and Th2 cells based on STAT4 expression, as STAT4-deficient mice display impaired Th1 development with enhanced Th2 cell development .

How should researchers design experiments to study STAT4 phosphorylation states using FITC-conjugated antibodies?

Designing experiments to study STAT4 phosphorylation requires careful planning to detect both tyrosine and serine phosphorylation, which regulate different aspects of STAT4 function:

• Cell stimulation conditions:

  • For tyrosine phosphorylation: Stimulate cells with IL-12 which activates JAK2 to phosphorylate STAT4 at tyrosine residues .

  • For serine phosphorylation (S721): Activate the MKK6/p38 pathway using appropriate stimuli or through expression of GADD45-β and GADD45-γ .

• Time course considerations: Include multiple time points (5, 15, 30, 60 minutes) after stimulation to capture the dynamic nature of phosphorylation events .

• Antibody selection: Use phospho-specific STAT4 antibodies (such as Phospho-STAT4 Ser721) alongside total STAT4 antibodies to determine the ratio of phosphorylated to total protein .

• Visualization techniques: For microscopy, co-staining with nuclear markers helps distinguish between cytoplasmic and nuclear localized phospho-STAT4, which is critical for understanding its transcriptional activity .

• Functional validation: Consider complementary functional assays such as reporter gene assays or cytokine production measurements (IFN-γ) to correlate phosphorylation status with functional outcomes .

This experimental approach allows researchers to dissect the differential roles of tyrosine versus serine phosphorylation, with the latter being particularly critical for IFN-γ production but not cell proliferation .

What are the key methodological considerations for immunofluorescence applications of FITC-conjugated STAT4 antibodies?

For successful immunofluorescence applications using FITC-conjugated STAT4 antibodies, researchers should consider these methodological aspects:

• Fixation protocol: Use 4% paraformaldehyde for 15-20 minutes at room temperature to preserve both protein localization and epitope accessibility .

• Permeabilization method: For nuclear transcription factors like STAT4, use 0.1-0.3% Triton X-100 to ensure nuclear penetration while preserving cellular architecture .

• Antibody dilution: Begin with the recommended range of 1:50-1:500 and optimize specifically for your cell type and fixation method . Higher concentrations may be needed for tissues compared to cell lines.

• Blocking conditions: Use 5-10% normal serum (from the species in which the secondary antibody was raised) with 1% BSA to minimize background signal .

• Counterstaining: Include nuclear staining (DAPI) and consider additional markers for co-localization studies:

  • For Th1 cell identification: Co-stain with T-cell markers (CD4) and Th1 markers (T-bet)

  • For activation state: Consider co-staining with phosphorylated STAT4 (pSer721) antibodies

• Signal amplification: If FITC signal is weak, avoid multiple exposure to excitation light as FITC is prone to photobleaching. Consider mounting media with anti-fade agents .

• Controls: Include isotype controls and STAT4-deficient cells or tissues (when available) as negative controls .

Optimal conditions should be validated for each specific cell type, with particular attention to signal-to-noise ratio and specificity verification.

How can FITC-conjugated STAT4 antibodies be used to investigate the differential roles of STAT4 isoforms (α and β) in inflammatory disease models?

STAT4 isoforms (Stat4α and Stat4β) differentially regulate inflammatory cytokines and have distinct effects on autoimmune disease progression . To investigate these differences using FITC-conjugated STAT4 antibodies:

• Isoform discrimination strategy: Select antibodies that can distinguish between full-length Stat4α and the Stat4β splice variant which lacks 44 amino acids at the C-terminus . This may require isoform-specific antibodies or combining the FITC-conjugated STAT4 antibody with isoform-specific unlabeled antibodies in competition assays.

• Experimental disease models: Utilize established models such as Experimental Autoimmune Encephalomyelitis (EAE), where Stat4β expression exacerbates disease while Stat4α attenuates it .

• Cytokine correlation analysis: Design flow cytometry panels that simultaneously detect STAT4 isoforms and key cytokines:

  • Stat4β correlates with increased IFN-γ and IL-17 production

  • Stat4α correlates with increased IL-10 production

• Transgenic approaches: Analyze tissues from transgenic mice expressing either Stat4α or Stat4β on a Stat4-deficient background to isolate isoform-specific effects .

• Temporal analysis: Monitor the dynamic expression of STAT4 isoforms during disease progression using time-course experiments with consistent antibody staining protocols.

This approach allows researchers to understand how STAT4 isoforms differentially regulate inflammatory cytokines, providing insights into potential therapeutic targets for autoimmune diseases .

What strategies can be employed to simultaneously detect STAT4 activation alongside other STAT family members in complex signaling networks?

Simultaneous detection of multiple activated STAT family members provides crucial insights into complex cytokine signaling networks. Researchers can employ these strategies:

• Multiplexed flow cytometry: Combine FITC-conjugated STAT4 antibodies with spectrally compatible fluorophores conjugated to antibodies against other STAT family members :

  • STAT1 (critical for IFN signaling)

  • STAT3 (IL-6, IL-10 signaling)

  • STAT5 (IL-2, IL-7, IL-15 signaling)

  • STAT6 (IL-4, IL-13 signaling)

• Phosphorylation state analysis: Include phospho-specific antibodies to determine activation status of each STAT protein alongside total protein levels .

• Multiparametric single-cell analysis: Employ imaging flow cytometry or mass cytometry (CyTOF) to correlate STAT4 activation with multiple other parameters at the single-cell level.

• Sequential immunofluorescence: For tissue sections, consider sequential staining protocols with antibody stripping between rounds to overcome spectral limitations.

• Co-immunoprecipitation strategies: Use STAT4 antibodies for immunoprecipitation followed by detection of interacting STAT proteins to identify heterodimer formation under various cytokine stimulation conditions .

• Stimulation matrix approach: Create a cytokine stimulation matrix (IL-12, IL-23, IFN-α/β, IL-2, IL-35) and monitor the resulting STAT activation patterns to delineate pathway-specific responses .

This comprehensive approach enables researchers to decipher the complex interplay between STAT4 and other STAT family members in response to different cytokine stimulations, providing insights into signaling network dynamics in normal and pathological conditions.

How can researchers optimize detection of nuclear translocation of STAT4 following cytokine stimulation?

Nuclear translocation of STAT4 is a critical step in its function as a transcription factor. To optimize detection of this process:

• Stimulation kinetics: Establish a precise time course for STAT4 nuclear translocation following cytokine stimulation :

  • IL-12 stimulation: Examine 15, 30, 60, and 120-minute timepoints

  • IFN-α/β stimulation: Include early timepoints (5-15 minutes) as translocation may be more rapid

• Subcellular fractionation: For biochemical approaches, optimize nuclear and cytoplasmic fractionation protocols with appropriate markers to confirm fraction purity:

  • Nuclear marker: Lamin B

  • Cytoplasmic marker: GAPDH or α-tubulin

• Live cell imaging: For dynamic studies, consider using cell lines expressing fluorescently-tagged STAT4 in combination with nuclear markers to track translocation in real-time.

• High-content imaging optimization:

  • Use automated imaging systems with nuclear segmentation algorithms

  • Quantify nuclear:cytoplasmic ratio of STAT4-FITC signal intensity

  • Apply standardized thresholds for defining "positive" nuclear translocation

• Confocal microscopy settings:

  • Use optical sectioning (z-stacks) to confirm genuine nuclear localization

  • Set optimal pinhole settings (1 Airy unit) for resolution of nuclear signals

  • Employ deconvolution algorithms to improve signal-to-noise ratio

• Inhibitor controls: Include controls with JAK inhibitors (to prevent STAT4 phosphorylation) and nuclear export inhibitors (like Leptomycin B) to validate the specificity of the nuclear translocation signal .

This methodological approach enables quantitative assessment of STAT4 nuclear translocation dynamics, a critical parameter for understanding its role in transcriptional regulation following cytokine stimulation.

How can FITC-conjugated STAT4 antibodies be utilized to study the role of STAT4 in autoimmune disease pathogenesis?

STAT4 plays a critical role in autoimmune disease pathogenesis through its regulation of inflammatory cytokines. Researchers can utilize FITC-conjugated STAT4 antibodies to investigate these mechanisms through:

• Patient-derived immune cell analysis: Compare STAT4 expression levels and phosphorylation status in peripheral blood mononuclear cells from autoimmune disease patients versus healthy controls using flow cytometry with standardized staining protocols .

• Tissue-specific expression patterns: Analyze STAT4 expression in affected tissues (e.g., synovium in rheumatoid arthritis, central nervous system in multiple sclerosis) using immunofluorescence microscopy with co-staining for cell type-specific markers .

• Genetic association correlation: In patients with STAT4 polymorphisms associated with autoimmune susceptibility, examine how these variants correlate with STAT4 protein expression and phosphorylation states .

• Therapeutic intervention monitoring: Evaluate changes in STAT4 expression and activation following treatment with cytokine-targeting biologics or JAK inhibitors to identify potential biomarkers of treatment response .

• Animal model applications: In models like EAE, use FITC-conjugated STAT4 antibodies to:

  • Track Th1/Th17 cell differentiation in lymphoid organs during disease induction

  • Monitor infiltrating immune cells in target tissues

  • Assess therapeutic efficacy of experimental treatments targeting the IL-12/STAT4 pathway

Studies have demonstrated that STAT4 isoforms differentially regulate inflammatory cytokines in association with distinct effects on autoimmune disease onset and severity, with Stat4β exacerbating EAE while Stat4α attenuates it . This highlights the importance of isoform-specific analysis in autoimmune disease research.

What are the technical considerations for using FITC-conjugated STAT4 antibodies in tissue samples from autoimmune disease models?

When analyzing tissue samples from autoimmune disease models using FITC-conjugated STAT4 antibodies, researchers should address these technical considerations:

• Tissue preparation protocols:

  • For frozen sections: Optimize fixation time (typically 10-15 minutes with 4% PFA) to preserve both tissue architecture and epitope accessibility

  • For paraffin-embedded tissues: Implement rigorous antigen retrieval (citrate buffer, pH 6.0, or EDTA buffer, pH 9.0) to expose STAT4 epitopes masked during fixation

• Background autofluorescence management:

  • For tissues with high autofluorescence (brain, spinal cord): Consider Sudan Black B treatment (0.1-0.3%) to quench lipofuscin autofluorescence

  • Use spectral unmixing when available to distinguish FITC signal from tissue autofluorescence

• Signal amplification strategies:

  • For low-expression tissues: Consider tyramide signal amplification systems compatible with FITC

  • Implement standardized exposure settings across experimental groups to allow quantitative comparisons

• Co-staining optimization:

  • Include cell-type specific markers to identify STAT4+ cells (CD4 for T cells, CD11c for dendritic cells)

  • Use nuclear counterstains (DAPI) to assess nuclear localization of STAT4 during active signaling

• Quantification approaches:

  • Develop consistent cell counting strategies across tissue sections

  • Use automated image analysis with standardized thresholds for STAT4 positivity

  • Report data as percentage of STAT4+ cells within defined cell populations

• Controls and validation:

  • Include tissues from STAT4-deficient animals as negative controls

  • Validate findings with alternative detection methods (e.g., chromogenic IHC) when possible

These considerations ensure reliable and reproducible detection of STAT4 in complex tissue environments characteristic of autoimmune disease models.

How does STAT4 serine phosphorylation affect T-cell differentiation and cytokine production, and how can this be monitored using FITC-conjugated antibodies?

STAT4 serine phosphorylation, particularly at position S721, plays a critical role in regulating T-cell differentiation and cytokine production. This phosphorylation event can be monitored using specialized approaches:

• Functional significance: Serine phosphorylation of STAT4 at S721 is critical for:

  • IL-12-induced IFN-γ production but not for cell proliferation

  • Optimal Th1 differentiation and immune response against intracellular pathogens

  • Maximal transcriptional activity of STAT4

• Signaling pathway integration: The MKK6/p38 pathway regulates STAT4 S721 phosphorylation through GADD45-β and GADD45-γ, linking STAT4 to stress response pathways . This represents a critical integration point between cytokine and stress signaling in immune cells.

• Methodological approach for monitoring:

  • Antibody selection: Use phospho-specific antibodies that recognize STAT4 phosphorylated at S721 along with total STAT4 antibodies

  • Flow cytometry protocol: Implement phospho-flow cytometry with careful attention to fixation techniques that preserve phospho-epitopes (methanol-based fixation often works better than formaldehyde for phospho-proteins)

  • Stimulation conditions: Compare IL-12 stimulation alone versus conditions that activate both JAK/STAT and p38 MAPK pathways

• Complementary functional readouts:

  • IFN-γ production measured by intracellular cytokine staining

  • T-bet expression as a marker of Th1 differentiation

  • Proliferation assays to distinguish growth effects from cytokine production

• Genetic validation approach: Utilize retroviral reconstitution of STAT4-deficient cells with either wild-type STAT4 or S721A mutant STAT4 to directly assess the role of this phosphorylation site . This approach has demonstrated that wild-type STAT4, but not the S721A mutant, restores normal Th1 differentiation and IFN-γ synthesis .

By implementing these approaches, researchers can gain insights into how post-translational modifications of STAT4 regulate T-cell differentiation and effector functions, potentially identifying novel therapeutic targets for immune-mediated diseases.

What are common pitfalls in FITC-conjugated STAT4 antibody experiments and how can they be addressed?

When working with FITC-conjugated STAT4 antibodies, researchers frequently encounter several technical challenges that can be systematically addressed:

• Signal fading/photobleaching:

  • Problem: FITC is susceptible to photobleaching during prolonged imaging sessions

  • Solution: Use anti-fade mounting media, minimize exposure to excitation light, consider image acquisition from unexposed fields of view, and store slides at -20°C protected from light

• High background fluorescence:

  • Problem: Non-specific binding or inadequate blocking

  • Solution: Optimize blocking (5-10% normal serum plus 1% BSA), include detergent in wash buffers (0.05-0.1% Tween-20), and validate specificity with STAT4-deficient controls

• Cell type-specific fixation issues:

  • Problem: Different cell types may require different fixation protocols for optimal staining

  • Solution: Compare multiple fixation methods (4% PFA, methanol, acetone) with your specific cell type; generally, 15-20 minutes with 4% PFA works well for most applications

• Nuclear versus cytoplasmic signal discrimination:

  • Problem: Difficulty distinguishing cytoplasmic from nuclear STAT4 staining

  • Solution: Use confocal microscopy with z-stack acquisition, include nuclear counterstains, and consider nuclear/cytoplasmic fractionation as a complementary approach

• Low signal intensity:

  • Problem: Insufficient detection of STAT4, particularly in cells with low expression

  • Solution: Optimize antibody concentration (testing a range from 1:50 to 1:500), consider signal amplification systems, and ensure epitope accessibility through appropriate permeabilization

• Inconsistent staining across experiments:

  • Problem: Batch-to-batch variation in staining intensity

  • Solution: Prepare aliquots of antibody to avoid freeze-thaw cycles, include standardization controls in each experiment, and maintain consistent protocols across experiments

Addressing these common pitfalls systematically will improve the reliability and reproducibility of experiments using FITC-conjugated STAT4 antibodies.

How can researchers validate the specificity of FITC-conjugated STAT4 antibodies in their experimental systems?

Validating antibody specificity is crucial for generating reliable research data. For FITC-conjugated STAT4 antibodies, researchers should implement these validation strategies:

• Genetic controls:

  • Gold standard: Test antibody in STAT4-knockout or STAT4-deficient cells/tissues which should show no specific staining

  • Knockdown validation: Compare staining in cells treated with STAT4-specific siRNA/shRNA versus control siRNA/shRNA

• Peptide competition assays:

  • Pre-incubate the antibody with excess immunizing peptide before staining

  • Specific staining should be blocked by the peptide while non-specific background remains

• Cross-validation with independent antibodies:

  • Compare staining patterns using antibodies from different sources or those recognizing different STAT4 epitopes

  • Consistent patterns across multiple antibodies increase confidence in specificity

• Molecular weight verification:

  • Perform western blot analysis in parallel to confirm the antibody recognizes a protein of the expected molecular weight (86 kDa for STAT4)

  • Look for expected molecular weight shifts with phosphorylated forms

• Expected expression pattern verification:

  • Confirm that staining is observed in tissues known to express STAT4 (thymus, spleen) but not in tissues that lack expression

  • Verify cell-type specific expression patterns match known STAT4 biology

• Signal intensity correlation with known biology:

  • Verify increased staining/signal in cells stimulated with IL-12 compared to unstimulated cells

  • Confirm nuclear translocation following appropriate cytokine stimulation

• Isotype control comparison:

  • Include a FITC-conjugated isotype control antibody (mouse IgG1 for many STAT4 antibodies) to distinguish non-specific binding

Implementing these validation approaches will ensure that experimental observations reflect genuine STAT4 biology rather than antibody artifacts.

What considerations are important when designing multiplexed flow cytometry panels that include FITC-conjugated STAT4 antibodies?

Designing effective multiplexed flow cytometry panels that include FITC-conjugated STAT4 antibodies requires careful consideration of several technical factors:

• Spectral compatibility planning:

  • FITC considerations: FITC (excitation ~495 nm, emission ~519 nm) has significant spectral overlap with PE (particularly problematic) and other green fluorophores

  • Recommended pairings: Combine FITC with fluorophores like APC, APC-Cy7, BV421, BV605, and PE-Cy7 to minimize compensation requirements

  • Compensation controls: Prepare single-color controls for accurate compensation matrix calculation

• Abundance-brightness matching:

  • Strategic assignment: Reserve brighter fluorophores (PE, APC) for low-abundance markers and consider FITC (medium brightness) for STAT4 when it's expressed at moderate levels

  • Expression level assessment: In cell types where STAT4 expression is low, consider using a brighter fluorophore-conjugated STAT4 antibody instead of FITC

• Panel design for STAT4 biology:

  • Essential markers to include:

    • Lineage markers: CD3, CD4 (T cells), CD19 (B cells), CD56 (NK cells)

    • Activation markers: CD25, CD69

    • Transcription factors: T-bet (Th1), GATA3 (Th2)

    • Cytokines: IFN-γ, IL-4 (for functional correlation)

  • Phospho-specific considerations: Include phospho-STAT4 (pY693, pS721) antibodies for activation status

• Fixation and permeabilization optimization:

  • Protocol selection: Transcription factor staining buffers (containing methanol or formaldehyde plus detergent) are required for STAT4 detection

  • Sequential staining: Consider staining surface markers before fixation/permeabilization for optimal results

  • Buffer compatibility: Ensure all antibodies in the panel perform well in the selected permeabilization buffer

• Sample preparation considerations:

  • Cell concentration: Maintain consistent cell concentrations (~1×10^6 cells/100 μl) across samples

  • Viability dye: Include a fixable viability dye compatible with FITC (far-red viability dyes are preferable)

  • Fc block: Include Fc receptor blocking to prevent non-specific binding, particularly in samples with high FcR expression

• Assay standardization:

  • Antibody titration: Determine optimal concentration of FITC-STAT4 antibody (typically 0.80 μg per 10^6 cells) through titration experiments

  • Internal controls: Include reference samples across experiments to normalize for day-to-day variations

  • Application-specific considerations: For phospho-flow, standardize stimulation conditions and time points

Careful attention to these considerations will result in robust multiplexed flow cytometry panels that generate reliable and interpretable data on STAT4 expression and function.

What are the future directions for STAT4 research using fluorescently labeled antibodies?

The field of STAT4 research utilizing fluorescently labeled antibodies is poised for significant advancements in several key areas:

• Single-cell multi-omics integration: Future research will likely combine FITC-conjugated STAT4 antibody staining with single-cell transcriptomics or proteomics to correlate STAT4 protein levels and activation states with global gene expression or protein profiles. This approach will provide unprecedented insights into how STAT4 regulates cell-specific responses to cytokine stimulation .

• Advanced imaging technologies: Emerging super-resolution microscopy techniques will enable visualization of STAT4 molecular interactions at nanometer resolution, potentially revealing previously undetected spatial organization of STAT4 signaling complexes within the nucleus and cytoplasm .

• Isoform-specific targeting: Development of antibodies with enhanced specificity for STAT4 isoforms (Stat4α and Stat4β) will facilitate more precise studies of their differential roles in inflammatory responses and autoimmune disease pathogenesis .

• Therapeutic monitoring applications: FITC-conjugated STAT4 antibodies will increasingly be used to monitor the efficacy of targeted therapies that modulate the IL-12/STAT4 pathway, potentially serving as biomarkers for treatment response in autoimmune and inflammatory conditions .

• Mass cytometry and spectral flow cytometry applications: Next-generation cytometry platforms will enable simultaneous detection of STAT4 alongside dozens of other proteins, providing comprehensive analysis of signaling networks in rare cell populations .

These advancements will collectively deepen our understanding of STAT4 biology and its role in immune regulation, potentially leading to novel therapeutic approaches for diseases involving dysregulated STAT4 signaling.

How might recent technological advances in fluorescence detection systems impact STAT4 antibody applications?

Recent technological advances in fluorescence detection systems are transforming STAT4 antibody applications in several significant ways:

• Spectral flow cytometry: Unlike conventional flow cytometry, spectral systems capture the complete emission spectrum of each fluorophore, enabling:

  • Improved separation of FITC from spectrally similar fluorophores

  • More comprehensive multiplexing (30+ parameters simultaneously)

  • Better resolution of STAT4 expression in heterogeneous cell populations with autofluorescence

• High-throughput imaging cytometry: Systems that combine flow cytometry with microscopy allow:

  • Visualization of STAT4 nuclear translocation at the single-cell level

  • Quantification of co-localization with other transcription factors

  • Higher throughput than conventional microscopy with retained spatial information

• Advanced tissue imaging platforms:

  • Multiplexed immunofluorescence systems using cyclic staining or spectral unmixing enable simultaneous visualization of STAT4 with 20+ other markers in tissue sections

  • Spatial transcriptomics combined with protein detection correlates STAT4 protein localization with gene expression profiles in the tissue microenvironment

• Improved fluorophore chemistry:

  • Next-generation fluorophores with enhanced brightness and photostability are replacing conventional FITC

  • These advances enable longer imaging sessions and improved detection of low-abundance STAT4 in challenging samples

• Artificial intelligence image analysis:

  • Machine learning algorithms now enable automated quantification of nuclear versus cytoplasmic STAT4 localization

  • Deep learning approaches can identify cell subtypes based on complex marker patterns including STAT4 expression

These technological advances collectively enhance the sensitivity, specificity, and information content derived from STAT4 antibody staining, driving new discoveries in STAT4 biology and its role in health and disease.