OLFML3 Antibody, FITC conjugated

What is OLFML3 and why is it an important target for fluorescence-based detection methods?

OLFML3 belongs to the olfactomedin (OLF) protein family and has been identified as a significant immunomodulatory protein with both intracellular and extracellular functions. Recent research has revealed OLFML3's roles in anti-viral immunity, tumorigenesis, and bacterial infection responses . Importantly, OLFML3 demonstrates regulatory functions in macrophage phagocytosis and migration, and plays a critical role in preventing lipopolysaccharide (LPS)-induced mitochondrial dysfunction . Its dual localization patterns (both secreted and intracellular) make it particularly suitable for fluorescence-based detection using FITC-conjugated antibodies, which can help researchers distinguish between different cellular pools of the protein.

What cellular compartments can be visualized using OLFML3-FITC antibodies?

OLFML3-FITC antibodies can be used to detect multiple cellular localizations. Research has demonstrated that despite containing a secretion signal peptide, OLFML3 functions both extracellularly and intracellularly . Specifically, OLFML3 has been located on the outer membrane of mitochondria through subcellular fractionation and immunofluorescence studies . When designing experiments with OLFML3-FITC antibodies, researchers should establish protocols that can differentiate between mitochondrial-associated, cytoplasmic, and secreted forms of OLFML3 using appropriate permeabilization techniques and co-staining markers.

How can researchers optimize immunofluorescence protocols for OLFML3-FITC antibodies?

Optimized immunofluorescence protocols for OLFML3-FITC antibodies should consider:

• Fixation method: Paraformaldehyde (4%) fixation preserves protein localization while maintaining fluorophore activity

• Permeabilization: Use Triton X-100 (0.1%) for total cellular staining; gentler detergents like saponin (0.01-0.05%) for selective membrane permeabilization to distinguish between intracellular and extracellular OLFML3

• Blocking: BSA (3-5%) with normal serum from the secondary antibody host species

• Antibody dilution: Begin with manufacturer's recommendations, then optimize based on signal-to-noise ratio

• Co-staining considerations: For mitochondrial co-localization, use markers such as TOM20 or MitoTracker dyes, as OLFML3 has been shown to localize to the outer mitochondrial membrane

How can researchers design experiments to investigate OLFML3-IRG1 interactions using fluorescence techniques?

To investigate OLFML3-IRG1 interactions, researchers should consider the following methodological approach:

• Co-immunoprecipitation with fluorescence verification: The interaction between OLFML3 and IRG1 has been confirmed through co-immunoprecipitation studies, where the OLF domain of OLFML3 was found to interact primarily with the C-terminal α+β domain of IRG1 . This can be followed by fluorescence-based detection.

• Proximity ligation assay (PLA): This technique can visualize protein-protein interactions in situ when the interacting proteins are within 40nm of each other. PLA combined with FITC-conjugated OLFML3 antibodies can provide spatial resolution of where OLFML3-IRG1 interactions occur.

• FRET analysis: Using FITC-conjugated OLFML3 antibodies alongside a compatible acceptor fluorophore-conjugated IRG1 antibody.

• Mitochondrial co-localization studies: As both OLFML3 and IRG1 have been shown to localize to mitochondria upon LPS stimulation, dual immunofluorescence staining combined with mitochondrial markers can help track this dynamic process .

What controls are essential when using OLFML3-FITC antibodies in inflammatory disease models?

When working with inflammatory disease models such as lipopolysaccharide (LPS)-induced acute lung injury (ALI), essential controls include:

• Isotype controls: Matched isotype control antibodies conjugated to FITC to determine background fluorescence and non-specific binding

• Knockout validation: Olfml3 knockout cells or tissues as negative controls, as demonstrated in studies where Olfml3 knockout reduced survival in LPS-induced ALI mouse models

• Temporal controls: Time-course experiments to capture dynamic changes in OLFML3 expression and localization, particularly following LPS stimulation where OLFML3 demonstrates time-dependent changes in localization

• Competing peptide controls: Pre-incubation of OLFML3-FITC antibodies with recombinant OLFML3 to validate specificity

• Dual staining validation: Co-staining with antibodies against known OLFML3 interaction partners (IRG1, AIFM1) to confirm specificity and co-localization patterns

How can OLFML3-FITC antibodies be used to track mitochondrial dysfunction in macrophages?

OLFML3-FITC antibodies can be effectively used to investigate mitochondrial dysfunction in macrophages through the following methodological approaches:

• Co-staining with mitochondrial functional markers: Combined staining with JC-1 probe to assess mitochondrial membrane potential (MMP, ΔΨm) alongside OLFML3 localization. Research has demonstrated that Olfml3 knockout reduces MMP in LPS-stimulated macrophages .

• Detection of mitochondrial OLFML3 translocation: Time-course analysis of OLFML3 translocation to mitochondria following LPS stimulation using fractionation and immunofluorescence approaches .

• Multiplex imaging: OLFML3-FITC antibodies can be combined with markers for mitochondrial reactive oxygen species (mtROS) and mitochondrial morphology to correlate OLFML3 localization with mitochondrial functional status .

• Quantitative analysis protocol:

  • Measure co-localization coefficients between OLFML3-FITC and mitochondrial markers

  • Correlate OLFML3 mitochondrial localization with mtROS levels

  • Track temporal dynamics of OLFML3 translocation during inflammation resolution

How can researchers investigate the role of OLFML3 in facilitating IRG1 mitochondrial localization?

Research has demonstrated that OLFML3 facilitates IRG1 mitochondrial localization through interaction with the mitochondrial transport protein AIFM1 . To investigate this pathway:

• Triple co-localization analysis: Employ OLFML3-FITC antibodies alongside differentially labeled IRG1 and AIFM1 antibodies to visualize the ternary complex formation. Research has confirmed that higher molecular weight complexes containing all three proteins form under LPS stimulation .

• Structured illumination microscopy: Use super-resolution techniques to precisely localize OLFML3 on the outer mitochondrial membrane and its spatial relationship with IRG1 and AIFM1.

• Domain-specific blocking: Utilize antibodies or peptides targeting specific domains of OLFML3 (OLF domain interacts with IRG1; CC domain interacts with AIFM1) to disrupt specific protein-protein interactions and observe effects on mitochondrial localization .

• Fractionation coupled with immunofluorescence: Combine subcellular fractionation with immunofluorescence to quantify the proportion of IRG1 localized to mitochondria in the presence or absence of OLFML3 .

What approaches should be used to study OLFML3's dual intracellular and extracellular functions?

OLFML3 presents a unique research challenge due to its dual functioning in both intracellular and extracellular environments . Methodological approaches should include:

• Differential permeabilization protocols: Utilize selective permeabilization protocols to distinguish between cell surface/extracellular and intracellular pools of OLFML3.

• Secretion inhibition studies: Combine brefeldin A treatment with OLFML3-FITC staining to distinguish between secreted and intracellular functions.

• Domain-specific antibodies: Signal peptide deletion experiments have shown that removal of the secretion signal does not affect OLFML3's subcellular localization or interaction with IRG1 . Researchers should utilize domain-specific antibodies to track different functional pools of OLFML3.

• Recombinant protein studies: Compare the effects of exogenously added recombinant OLFML3 versus endogenously expressed protein on cellular functions such as macrophage phagocytosis and migration .

• Conditioned media transfer experiments: Use FITC-labeled antibodies to track OLFML3 internalization from conditioned media to distinguish extracellular signaling from intracellular functions.

How can OLFML3-FITC antibodies be utilized in tumor microenvironment studies?

Clinical studies have correlated high OLFML3 expression with reduced disease-free survival in colorectal cancer patients . OLFML3-FITC antibodies can be used to:

• Characterize tumor-associated macrophage (TAM) populations: Studies have shown that antibody-mediated blockade of OLFML3 decreases recruitment of tumor-promoting TAMs . FITC-conjugated antibodies can help identify OLFML3-expressing cells within the tumor microenvironment.

• Monitor therapeutic responses: Track changes in OLFML3 expression patterns following anti-OLFML3 and anti-PD-1 combination therapy, which has shown enhanced antitumor efficacy compared to monotherapy .

• Multiplex immunofluorescence panels: Combine OLFML3-FITC with markers for:

  • TAMs (CD68, CD163)

  • NKT cells (NK1.1, CD3)

  • Tumor vasculature (CD31)

  • Immune checkpoint proteins (PD-1, PD-L1)

• In vivo imaging: For animal models, consider intravital microscopy using OLFML3-FITC antibodies to track dynamic changes in OLFML3 expression during tumor progression and therapy.

How should researchers address discrepancies between OLFML3 antibody staining patterns and expected localization?

When OLFML3-FITC antibody staining patterns differ from expected localizations, researchers should:

• Validate antibody specificity: Confirm specificity using Olfml3 knockout cells as negative controls, as demonstrated in studies that validated phenotypic changes by Olfml3 knockout .

• Consider fixation artifacts: Different fixation methods can alter protein localization. Compare paraformaldehyde, methanol, and glutaraldehyde fixation to determine optimal preservation of OLFML3 localization.

• Evaluate cell activation state: OLFML3 localization is dynamic and stimulus-dependent. For example, LPS stimulation induces mitochondrial localization of OLFML3 . Carefully document cell activation states when interpreting staining patterns.

• Check for protein-protein interactions: OLFML3 interacts with multiple partners including IRG1 and AIFM1 . These interactions may mask antibody epitopes or alter apparent localization.

• Combine detection methods: Cross-validate fluorescence data with subcellular fractionation, as demonstrated in studies that combined immunofluorescence with mitochondrial fractionation to confirm OLFML3 localization .

What factors might affect the sensitivity and specificity of OLFML3-FITC antibody detection in different experimental contexts?

Several factors can impact OLFML3-FITC antibody performance:

• Protein expression levels: OLFML3 expression varies across cell types and in response to stimuli like LPS. Lower expression may require signal amplification techniques.

• Complex formation: OLFML3 forms high molecular weight complexes (>400kD) with IRG1 and AIFM1 , which may affect epitope accessibility.

• Post-translational modifications: Consider whether phosphorylation or other modifications might affect antibody binding, especially under different cellular activation states.

• Autofluorescence considerations: Macrophages, which highly express OLFML3, often exhibit significant autofluorescence that can interfere with FITC detection. Implement appropriate controls and consider alternative fluorophores if needed.

• Photobleaching effects: FITC is susceptible to photobleaching. Implement anti-fade reagents and minimize exposure times when designing long-term imaging experiments.

How might OLFML3 antibodies be utilized to develop novel therapeutic approaches for inflammatory diseases?

Research has demonstrated that OLFML3 plays a protective role in lipopolysaccharide (LPS)- and Pseudomonas aeruginosa-induced acute lung injury (ALI) mouse models . Future therapeutic applications using OLFML3 antibodies might include:

• Targeted delivery systems: Development of OLFML3 antibody-conjugated nanoparticles for delivering anti-inflammatory compounds specifically to macrophages.

• Monitoring therapeutic efficacy: Using FITC-conjugated OLFML3 antibodies to track changes in OLFML3 expression and localization during treatment of inflammatory conditions.

• Combined immunotherapy approaches: Based on findings that targeting OLFML3 increased the antitumor efficacy of anti-PD-1 checkpoint inhibitor therapy , explore other combination approaches in inflammatory diseases.

• Biomarker development: Utilizing OLFML3 antibodies for detecting changes in OLFML3 expression as a biomarker for disease progression or treatment response in inflammatory conditions.

• Mitochondrial function modulation: Given OLFML3's role in preventing mitochondrial dysfunction, explore therapeutic approaches targeting the OLFML3-IRG1-AIFM1 pathway to maintain mitochondrial homeostasis during inflammatory responses .

What emerging technologies might enhance the utility of OLFML3-FITC antibodies in basic and translational research?

Emerging technologies that could enhance OLFML3-FITC antibody applications include:

• Single-cell proteomics: Combining OLFML3-FITC detection with single-cell mass cytometry to characterize heterogeneity in OLFML3 expression and function across cell populations.

• CRISPR-based tracking: Combining CRISPR-mediated tagging of endogenous OLFML3 with FITC-antibody detection for live-cell tracking of OLFML3 dynamics.

• Super-resolution microscopy: Techniques like STORM or PALM could provide nanometer-scale resolution of OLFML3 localization on the outer mitochondrial membrane and its interactions with IRG1 and AIFM1 .

• Organ-on-chip models: Utilizing OLFML3-FITC antibodies in microfluidic organ-on-chip platforms to study OLFML3 function in more physiologically relevant contexts.

• AI-assisted image analysis: Implementing machine learning algorithms to quantify subtle changes in OLFML3 localization patterns in response to different stimuli or disease states.