OAS3 Antibody, FITC conjugated

What is OAS3 and what role does it play in cellular immune responses?

OAS3 (2'-5'-oligoadenylate synthase 3) is an interferon-induced, dsRNA-activated antiviral enzyme critical in cellular innate antiviral response. It functions by synthesizing preferentially dimers of 2'-5'-oligoadenylates (2-5A) from ATP which then bind to the inactive monomeric form of ribonuclease L (RNase L), leading to its dimerization and subsequent activation. This activation results in degradation of cellular and viral RNA, inhibiting protein synthesis and terminating viral replication. Beyond its antiviral role, OAS3 participates in other cellular processes including apoptosis, cell growth, differentiation and gene regulation . OAS3 has demonstrated specific antiviral activity against multiple viruses including Chikungunya virus (CHIKV), Dengue virus, Sindbis virus (SINV) and Semliki forest virus (SFV) .

What are the standard applications for OAS3 Antibody, FITC conjugated in research?

OAS3 Antibody with FITC conjugation is primarily used in immunofluorescence (IF) techniques and flow cytometry. Most commercial preparations indicate validated applications including ELISA and Dot Blot . The FITC conjugation (excitation/emission: 499/515 nm) makes this antibody particularly suitable for direct immunofluorescence studies without requiring secondary antibody detection systems . Different manufacturers recommend varying dilutions, typically 1:50-1:200 for IF applications . While some vendors have validated the antibody for additional applications, researchers should conduct their own validation for specific experimental contexts.

How does FITC conjugation affect antibody performance compared to unconjugated versions?

FITC conjugation provides direct visualization capabilities but may affect antibody binding characteristics in several ways:

• The conjugation process may sterically hinder epitope recognition if fluorophores are attached near the antigen-binding domain

• The fluorophore:protein ratio is critical - over-conjugation can reduce antigen binding while under-conjugation produces weak signals

• FITC-conjugated antibodies may show slightly reduced binding affinity compared to unconjugated counterparts due to modification of amino acid residues during the conjugation process

• FITC-conjugated antibodies are more sensitive to photobleaching than certain other fluorophores

When transitioning from unconjugated to FITC-conjugated antibodies, researchers should validate optimal working dilutions empirically, as the recommended dilution may differ significantly from unconjugated versions (typically requiring higher concentrations of the conjugated antibody) .

What are the optimal storage conditions for maintaining FITC-conjugated OAS3 antibody activity?

For maximum stability and fluorescence retention, OAS3 Antibody with FITC conjugation should be stored according to these guidelines:

• Store at -20°C or -80°C in aliquots to minimize freeze-thaw cycles

• Protect from light exposure during storage and handling to prevent photobleaching of the FITC fluorophore

• Store in manufacturer's recommended buffer, typically containing 50% glycerol, 0.01M PBS at pH 7.4, and 0.03% Proclin 300 as a preservative

• Avoid repeated freeze-thaw cycles as these can significantly compromise antibody binding and fluorescence intensity

• When thawed for use, maintain at 4°C and use within 2-3 days for optimal performance

Properly stored, most preparations maintain activity for at least 12 months, though gradual decline in fluorescence intensity may occur over extended storage periods .

How should researchers optimize OAS3 Antibody, FITC conjugated for immunofluorescence microscopy protocols?

Optimization of FITC-conjugated OAS3 antibody for immunofluorescence requires systematic evaluation of multiple parameters:

Protocol optimization strategies:

• Concentration titration: Test a range of dilutions between 1:20-1:200 as recommended by different manufacturers

• Antigen retrieval evaluation: For fixed tissues, citrate buffer (pH 6.0) or TE buffer (pH 9.0) may improve epitope accessibility

• Blocking optimization: Use 5-10% normal serum from the same species as the secondary antibody with 0.1-0.3% Triton X-100 for permeabilization

• Incubation time and temperature: Compare short incubations (1-2 hours) at room temperature versus overnight at 4°C

• Control experiments: Include a no-primary antibody control and isotype control to assess non-specific binding

• Signal amplification: For weak signals, consider implementing tyramide signal amplification techniques while maintaining specificity

The optimal protocol will require empirical determination for each tissue type or cell line .

What are the most effective strategies for troubleshooting high background or weak signals when using FITC-conjugated OAS3 antibody?

When encountering technical issues with FITC-conjugated OAS3 antibody, systematic troubleshooting approaches include:

For high background:

• Increase blocking time (2-3 hours) and concentration (5-10% normal serum)

• Add 0.1-0.3% BSA to antibody diluent

• Reduce primary antibody concentration

• Include 0.1% Tween-20 in wash buffers and increase washing steps

• Examine for tissue autofluorescence; consider Sudan Black B treatment (0.1-0.3%) to reduce autofluorescence

For weak signals:

• Optimize antigen retrieval methods (test both heat-induced and enzymatic methods)

• Increase antibody concentration within recommended range

• Extend incubation time (overnight at 4°C)

• Ensure proper storage to maintain FITC fluorescence intensity

• Use higher-sensitivity detection systems or consider signal amplification techniques

Each troubleshooting approach should be tested systematically, changing only one variable at a time to determine the specific cause of the issue .

How can OAS3 Antibody, FITC conjugated be utilized in flow cytometry for studying cellular immune responses?

For flow cytometry applications with FITC-conjugated OAS3 antibody, researchers should implement these specialized protocols:

• Cell preparation: Use gentle fixation (2% paraformaldehyde for 10-15 minutes) followed by permeabilization (0.1% saponin or 0.1% Triton X-100) since OAS3 is primarily intracellular

• Antibody titration: Determine optimal staining concentration using a titration series (typically starting at 1:50-1:200 dilution)

• Compensation setup: Include single-color controls to compensate for spectral overlap with other fluorophores in the 488nm excitation channel

• Gating strategy: Implement hierarchical gating beginning with FSC/SSC to identify cells of interest, followed by viability marker exclusion before analyzing OAS3 expression

• Controls: Include FMO (Fluorescence Minus One) controls, isotype controls, and biological negative/positive controls

For detecting OAS3 in different immune cell populations, consider dual staining with lineage markers (e.g., CD14 for monocytes, CD3 for T cells) to correlate OAS3 expression with specific cell types. This approach is particularly valuable when studying IFN-primed macrophages that exhibit high OAS3 expression .

What are the considerations for using OAS3 Antibody, FITC conjugated in multiplex immunofluorescence studies?

When designing multiplex immunofluorescence panels including FITC-conjugated OAS3 antibody:

• Panel design: FITC (excitation/emission: 499/515 nm) is compatible with multiplexing but has potential spectral overlap with other 488nm-excited fluorophores. Careful panel design should account for:

  • Fluorophore brightness hierarchy (FITC has moderate brightness)

  • Antigen expression levels (pair brightest fluorophores with lowest-expressed antigens)

  • Spatial distribution of antigens (avoid fluorophores with similar emission spectra for co-localized antigens)

• Sequential staining: Consider sequential rather than cocktail staining when using multiple rabbit-derived antibodies

• Autofluorescence mitigation: Implement tissue-specific autofluorescence reduction methods:

  • Photobleaching

  • Sudan Black B treatment (0.1-0.3%)

  • Autofluorescence quenching reagents

• Spectral unmixing: Use spectral imaging and computational unmixing algorithms for improved separation of fluorophores with overlapping spectra

• Cross-reactivity testing: Validate absence of cross-reactivity between antibodies in the multiplex panel

How can researchers validate the specificity of OAS3 Antibody, FITC conjugated in their experimental system?

Comprehensive validation of OAS3 antibody specificity requires multiple complementary approaches:

• Positive and negative controls:

  • Use IFN-α/β stimulated cells as positive controls (induces OAS3 expression)

  • Include OAS3 knockout or knockdown samples as negative controls

  • Test cell lines with known differential expression of OAS3 (e.g., A375, A431, HeLa cells show positive reactivity)

• Western blot correlation:

  • Confirm antibody detects expected molecular weight band (100-120 kDa)

  • Compare patterns between unconjugated and FITC-conjugated versions

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide to block specific binding

  • Compare staining with and without peptide competition

• Cross-reactivity assessment:

  • Test for potential cross-reactivity with other OAS family members (OAS1, OAS2, OASL)

  • Some sources note possible weak cross-reactivity with OAS1

• Orthogonal detection methods:

  • Correlate protein expression with mRNA expression data

  • Compare multiple antibodies targeting different epitopes of OAS3

How does OAS3 function differ from other members of the OAS family?

OAS3 exhibits distinct structural and functional characteristics compared to other OAS family members:

OAS3 synthesizes predominantly dimeric 2-5A oligomers unlike OAS2 which produces primarily trimeric and tetrameric 2-5A products. OAS3's higher affinity for dsRNA makes it particularly effective at low dsRNA concentrations. Additionally, OAS3's unique ability to mediate antiviral effects through both RNase L-dependent and independent pathways distinguishes it from other family members .

What recent advances have been made in understanding OAS3's role in cancer immunology?

Recent research has revealed unexpected roles for OAS3 in tumor immunology:

• M2d macrophage polarization: OAS3 has been identified as a key mediator in tumor-associated macrophage (TAM) polarization. Research published in 2024 found that:

  • Lactate derived from pancreatic cancer (PC) cells transfers to macrophages, upregulating OAS3 via m⁶A modification

  • This leads to M2d polarization characterized by increased IL-10 and VEGF-A secretion

  • M2d macrophages subsequently impair CD8⁺ T cell function

• Correlation with immunosuppressive markers: OAS3 expression positively correlates with:

  • Immunosuppressive cell markers (CD163, CD206)

  • Immune checkpoint molecules (PD-L1)

  • Increased Treg infiltration

  • T cell exhaustion markers

• Therapeutic targeting potential: Knockdown of OAS3 in macrophages:

  • Reduces PD-L1 expression on macrophages

  • Decreases IL-10 and VEGF-A secretion

  • Restores CD8⁺ T cell activity

  • Enhances efficacy of gemcitabine and anti-PD-L1 therapy in pancreatic cancer models

This emerging research positions OAS3 as a promising immunotherapeutic target, particularly for "immune-excluded" tumors characterized by abundant peritumoral stromal immune cells .

What are the methodological considerations for studying OAS3 in its activated versus non-activated states?

Studying OAS3 activation state requires specialized techniques that distinguish between basal and activated forms:

• Induction protocols for activated state:

  • IFN-α/β treatment (50-1000 IU/ml, 12-24 hours) induces OAS3 expression

  • Poly(I:C) transfection (0.5-1 μg/ml) activates existing OAS3

  • Viral infection models provide physiological activation

• Activity assays:

  • Radiometric assays measuring ATP→2-5A conversion

  • FRET-based reporter assays for monitoring RNase L activation

  • IP-kinase assays assessing phosphorylation status

• Structural detection of activation:

  • Proximity ligation assays to detect OAS3-dsRNA interactions

  • Co-immunoprecipitation studies examining protein-protein interactions in activated state

  • Conformational antibodies specifically recognizing activated OAS3

• Functional readouts:

  • RNase L activation (rRNA degradation patterns)

  • Downstream signaling cascades (PKR, MAPK pathways)

  • Cellular localization changes upon activation using the FITC-conjugated antibody

When designing experiments to distinguish activation states, researchers should include appropriate controls (IFN receptor knockout, OAS3 knockout) and time course analyses to capture the dynamic nature of OAS3 activation.

How might researchers utilize OAS3 antibodies to investigate the emerging role of OAS3 in cancer immunotherapy resistance?

Based on recent findings implicating OAS3 in immunosuppressive tumor microenvironments, researchers can design experiments using FITC-conjugated OAS3 antibodies to:

• Characterize OAS3⁺ cell populations in tumors:

  • Multiplex immunofluorescence with TAM markers (CD163, CD206) and OAS3

  • Flow cytometric quantification of OAS3 expression across immune cell subsets

  • Spatial distribution analysis of OAS3⁺ cells relative to CD8⁺ T cells and tumor cells

• Monitor therapeutic responses:

  • Evaluate OAS3 expression changes during immunotherapy treatment

  • Correlate OAS3 levels with response/resistance patterns

  • Develop prognostic scoring based on OAS3⁺ cell infiltration patterns

• Mechanistic studies:

  • Track OAS3 expression changes following metabolite exposure (lactate, etc.)

  • Investigate epigenetic regulation of OAS3 in different TME conditions

  • Examine OAS3-dependent secretome alterations in macrophages

• Therapeutic targeting validation:

  • Confirm target engagement of OAS3-directed therapeutics

  • Develop companion diagnostics for OAS3-targeting therapies

  • Screen for synthetic lethal interactions with OAS3 inhibition

Such approaches could establish OAS3 as a clinically relevant biomarker for immunotherapy response prediction and therapeutic targeting.

What techniques can be used to distinguish between OAS3's RNase L-dependent and RNase L-independent antiviral pathways using FITC-conjugated antibodies?

To dissect the dual antiviral mechanisms of OAS3, researchers can implement these advanced approaches:

• Genetic separation of pathways:

  • RNase L knockout systems to isolate RNase L-independent functions

  • OAS3 domain-specific mutants that selectively disrupt RNase L activation

  • Combination with FITC-conjugated OAS3 antibody to track localization changes

• Pathway-specific visualization:

  • Co-localization studies using FITC-OAS3 antibody with RNase L

  • Live-cell imaging to track temporal dynamics of pathway activation

  • Proximity ligation assays to detect specific protein-protein interactions

• Functional readouts:

  • RNase L-specific substrate degradation patterns

  • Transcriptomic profiling in RNase L-sufficient vs. deficient systems

  • Viral replication assays with pathway-specific inhibitors

• Structural biology approaches:

  • Antibody epitope mapping to identify conformation-specific states

  • Domain-specific antibodies to track structural changes during activation

By combining these approaches with the subcellular localization information provided by FITC-conjugated OAS3 antibodies, researchers can create comprehensive models of OAS3's dual antiviral mechanisms .

How can computational analysis be integrated with OAS3 immunofluorescence data to develop predictive models of antiviral responses?

Integrating computational approaches with OAS3 immunofluorescence data enables sophisticated analysis frameworks:

• Machine learning for image analysis:

  • Automated quantification of OAS3 expression patterns

  • Cell-type specific OAS3 localization classification

  • Correlation of expression patterns with functional outcomes

• Systems biology integration:

  • Multi-parameter correlation of OAS3 with other immune markers

  • Network analysis of OAS3-associated proteins

  • Dynamic modeling of OAS3 activation kinetics

• Biomarker development:

  • Identification of OAS3 expression thresholds predictive of antiviral responses

  • Multi-marker signatures incorporating OAS3 and related pathway components

  • Patient stratification algorithms based on OAS3 and response patterns

• Spatial analysis frameworks:

  • Neighborhood analysis of OAS3⁺ cells in tissue contexts

  • Interaction maps between OAS3⁺ cells and other immune populations

  • Tissue architecture influence on OAS3 pathway activation

This integrated approach transforms static immunofluorescence data into dynamic predictive models of antiviral efficacy, particularly valuable in assessing therapeutic responses to viral infections where OAS3 plays a critical role .

What are the most rigorous methods for validating lot-to-lot consistency of FITC-conjugated OAS3 antibodies?

Ensuring experimental reproducibility requires comprehensive lot validation protocols:

• Spectrophotometric assessment:

  • Absorbance ratios (280nm vs. 495nm) to determine F/P (fluorophore-to-protein) ratio

  • Consistent F/P ratios between 3-7 typically yield optimal performance

  • Emission spectra analysis to confirm fluorescence properties

• Performance validation:

  • Side-by-side testing with reference lot using identical protocols

  • Multi-parameter comparative analysis:

    • Signal intensity at standardized dilutions

    • Background levels under identical conditions

    • Signal-to-noise ratios across dilution series

• Application-specific validation:

  • Flow cytometry: Mean fluorescence intensity comparison

  • Immunofluorescence microscopy: Integrated density measurements

  • Dot blot: Signal intensity at serial dilutions

• Stability testing:

  • Accelerated stability testing under defined conditions

  • Photobleaching resistance comparison between lots

  • Freeze-thaw stability assessment

• Identity confirmation:

  • Peptide competition assays to verify epitope specificity

  • Western blot performance to confirm molecular weight specificity

Implementing standardized lot testing protocols ensures experimental reproducibility and reliable data generation across studies.

How should researchers approach cross-platform validation when using OAS3 antibodies across different detection systems?

Cross-platform validation requires systematic comparison strategies:

• Sequential validation approach:

  • Begin with the most established application (typically Western blot)

  • Progress to more complex applications (IF, flow cytometry)

  • Document optimal conditions for each platform

• Concentration optimization:

  • Determine optimal working concentration independently for each platform

  • Typical dilution ranges:

    • ELISA: 1:1000-1:5000

    • Immunofluorescence: 1:50-1:200

    • Flow cytometry: 1:50-1:100

• Buffer compatibility assessment:

  • Test performance in application-specific buffers

  • Evaluate impact of specific components (detergents, salts, blocking proteins)

• Control system development:

  • Establish platform-specific positive and negative controls

  • Create reference standards for quantitative comparisons

• Correlation analysis:

  • Quantitatively compare results between platforms

  • Assess concordance in detecting relative expression differences

  • Document platform-specific limitations or biases

Systematic cross-platform validation ensures consistent biological interpretations regardless of the detection method employed.

How can OAS3 expression analysis using FITC-conjugated antibodies contribute to personalized viral infection treatment strategies?

OAS3 expression profiling could enable precision medicine approaches for viral infections:

• Patient stratification:

  • Baseline OAS3 expression levels may predict interferon responsiveness

  • Genetic variants affecting OAS3 function can be correlated with expression patterns

  • OAS3 activation state assessment may identify patients with impaired antiviral pathways

• Treatment response monitoring:

  • Serial sampling to track OAS3 expression changes during antiviral therapy

  • Correlation with viral load and clinical outcomes

  • Early identification of non-responders based on OAS3 activation patterns

• Combination therapy rationales:

  • Patients with defective OAS3 pathways may benefit from alternative antiviral strategies

  • OAS3 expression patterns may guide adjuvant therapy selection

  • Immunomodulatory approaches could be personalized based on OAS3 functional status

• Biobanking and retrospective analysis:

  • Archived samples can be analyzed for OAS3 expression correlations with outcomes

  • Machine learning algorithms incorporating OAS3 data may identify novel response predictors

  • Population-level analyses may reveal demographic-specific OAS3 response patterns

By incorporating OAS3 expression profiling into clinical decision-making algorithms, more targeted and effective antiviral strategies can be developed.

What methodological considerations exist for studying the recently discovered role of OAS3 in tumor-associated macrophage function?

Investigating OAS3 in tumor-associated macrophages requires specialized approaches:

• Sample preparation considerations:

  • Fresh tissue processing within 2-4 hours to preserve macrophage phenotypes

  • Optimized disaggregation protocols to maintain surface marker integrity

  • Specialized fixation (light PFA fixation, 2-3%) to preserve both surface markers and intracellular OAS3

• Macrophage phenotyping strategies:

  • Multi-parameter flow cytometry panels including:

    • M1 markers: CD80, CD86, HLA-DR

    • M2 markers: CD163, CD206

    • M2d-specific: IL-10, VEGF, PD-L1

    • OAS3 (FITC-conjugated)

  • Spectral flow cytometry for comprehensive phenotyping

• Functional correlation assays:

  • OAS3⁺ macrophage isolation and functional testing

  • Secretome analysis correlated with OAS3 expression

  • T cell suppression assays stratified by OAS3 expression

• In situ analysis approaches:

  • Multiplex immunofluorescence to preserve spatial context

  • Digital spatial profiling for comprehensive marker analysis

  • Single-cell RNA-seq with protein (CITE-seq) to correlate OAS3 mRNA and protein

• Intervention studies:

  • siRNA-mediated OAS3 knockdown in isolated TAMs

  • Ex vivo drug screening on OAS3⁺ macrophages

  • In vivo targeting validation with macrophage-specific delivery systems

These methodological considerations enable robust investigation of OAS3's newly discovered role in tumor immunosuppression.

How might future developments in antibody technology enhance the utility of OAS3 detection in research and clinical applications?

Emerging antibody technologies hold promise for advancing OAS3 research:

• Next-generation fluorophores:

  • Quantum dot conjugation for enhanced photostability and brightness

  • Near-infrared fluorophores for reduced autofluorescence and deeper tissue imaging

  • Self-quenching antibodies that fluoresce only upon target binding

• Conformational state-specific antibodies:

  • Antibodies specifically recognizing activated vs. inactive OAS3

  • Phosphorylation state-specific antibodies for activation status

  • Conformation-sensitive nanobodies for minimally invasive tracking

• Multimodal imaging capabilities:

  • Dual-modality probes combining fluorescence with MRI or PET

  • Theranostic antibody conjugates for simultaneous imaging and targeting

  • Mass cytometry (CyTOF) compatible antibodies for highly multiplexed detection

• Intracellular delivery innovations:

  • Cell-penetrating antibody formats for live-cell imaging

  • Conditionally activatable antibodies for spatiotemporal control

  • mRNA-encoded antibody fragments for endogenous expression

• Clinical translation potential:

  • Humanized OAS3 antibodies for in vivo imaging

  • Companion diagnostic development for OAS3-targeted therapies

  • Standardized immunohistochemistry protocols for clinical implementation