S1PR2 Antibody, FITC conjugated

What is S1PR2 and what cellular functions does it regulate?

S1PR2 is a G protein-coupled receptor for the lysosphingolipid sphingosine 1-phosphate (S1P). It mediates diverse physiological effects across various cell types and tissues. S1PR2 plays crucial roles in cell proliferation and suppression of apoptosis when activated by S1P. Additionally, it serves as a receptor for the chemokine-like protein FAM19A5, mediating inhibitory effects on vascular smooth muscle cell proliferation and migration. S1PR2 primarily couples with G protein families including Gi/o, Gq/11, and G12/13, with in vivo studies showing strict dependence on Gα13 signaling in lymphocytes and endothelial cells.

What is the difference between S1PR2 and other S1P receptors?

S1PR2 differs from other S1P receptors (S1PR1-5) primarily in its G protein coupling preferences and downstream signaling pathways. While S1PR1 and S1PR5 couple preferentially to Gi/o proteins, S1PR2 and S1PR3 can couple to multiple G protein families including Gi/o, Gq/11, and G12/13. This contributes to their distinct cellular effects. Specifically, S1PR2 demonstrates strong coupling to G13, which plays a critical role in cerebrovascular permeability and integrity. Unlike S1PR1, which promotes lymphocyte egress from lymphoid organs, S1PR2 has been implicated in increasing vascular permeability and disruption of neurovascular integrity following ischemic injury.

What is the molecular structure of S1PR2 and how does it interact with G proteins?

The recently elucidated cryo-electron microscopy structure of S1P-bound S1PR2 coupled to heterotrimeric G13 reveals critical interaction mechanisms. A key structural feature involves the interaction between S1PR2's intracellular loop 2 (ICL2) and transmembrane helix 4, which positions ICL2 to engage with the α5 helix of Gα13. This structural arrangement is essential for complex assembly and signal transduction. Disease-associated mutations in S1PR2 can disrupt this receptor structure, affecting its signaling capacity. The structure also revealed that FTY720-P, known primarily as an agonist for other S1P receptors, can trigger G13 activation via S1PR2, with the S1PR2 F274I variant showing increased G13 activity with both FTY720-P and S1P.

What criteria should researchers consider when selecting an S1PR2 antibody for their experiments?

Researchers should evaluate several critical parameters when selecting an S1PR2 antibody:

• Epitope specificity: Verify the antibody targets a specific region of S1PR2 (e.g., ab235919 targets human S1PR2 within aa 250 to C-terminus). This is essential for distinguishing S1PR2 from other S1P receptors with structural similarity.

• Validated applications: Confirm the antibody has been validated for your intended application (e.g., Western blot, immunocytochemistry/immunofluorescence). Check whether the manufacturer has tested the specific species and application combination.

• Species reactivity: Ensure compatibility with your experimental model system (human, mouse, rat, etc.).

• Literature citations: Review publications that have successfully used the antibody for similar applications.

• Conjugation compatibility: For FITC-conjugated antibodies, verify the fluorophore doesn't interfere with the binding domain and check the excitation/emission spectra are compatible with your detection system.

How does FITC conjugation affect S1PR2 antibody performance?

FITC conjugation can influence S1PR2 antibody performance in several ways:

• Sensitivity and signal-to-noise ratio: FITC provides direct visualization without secondary antibodies, but may have lower signal intensity compared to some other fluorophores, potentially requiring signal amplification in tissues with low S1PR2 expression.

• Photobleaching considerations: FITC is more susceptible to photobleaching than some alternative fluorophores, requiring careful imaging protocols to maintain signal integrity during extended microscopy sessions.

• pH sensitivity: FITC fluorescence is optimal at alkaline pH (>7.5) and decreases significantly in acidic environments, which may affect detection in certain cellular compartments.

• Conjugation ratio: The fluorophore-to-protein ratio affects both brightness and potential interference with antigen recognition. Optimal ratios must be determined for each application.

When working with FITC-conjugated S1PR2 antibodies, researchers should include appropriate controls to account for potential autofluorescence, especially in brain tissue where lipofuscin can generate background signal in similar spectral ranges.

What are the optimal conditions for using FITC-conjugated S1PR2 antibodies in immunofluorescence microscopy?

For optimal immunofluorescence microscopy with FITC-conjugated S1PR2 antibodies:

• Fixation protocol: 4% paraformaldehyde (PFA) for 24 hours has been successfully used for brain tissue, followed by cryoprotection in 30% sucrose solution.

• Section thickness: 30 μm sections are appropriate for brain tissue visualization.

• Blocking solution: Use TBS-blocking solution containing 1% bovine serum albumin, 0.2% skim milk, and 0.3% Triton X-100 to reduce non-specific binding.

• Antibody dilution: Start with manufacturer's recommended dilution (typically 1:100 for primary S1PR2 antibodies) and optimize if necessary.

• Incubation conditions: Overnight incubation at 4°C on a shaker provides consistent results.

• Co-staining options: Co-staining with endothelial markers such as CD31 (1:100) helps identify vascular-specific S1PR2 expression.

• Nuclear counterstaining: Brief DAPI staining (7 minutes) provides nuclear context.

• Mounting medium: Use anti-fade mounting medium to preserve FITC fluorescence.

• Controls: Include tissue from S1PR2 knockout mice as negative controls to confirm antibody specificity.

How can FITC-conjugated S1PR2 antibodies be used to study cerebrovascular integrity?

FITC-conjugated S1PR2 antibodies provide valuable tools for investigating cerebrovascular integrity through several methodological approaches:

• Vessel-specific expression analysis: Co-localization studies with endothelial markers (CD31) can reveal S1PR2 expression patterns in cerebral microvessels under normal and pathological conditions.

• Temporal expression dynamics: Time-course immunofluorescence studies following ischemic or inflammatory challenges can track changes in vascular S1PR2 expression, which is upregulated in ischemic microvessels.

• Cellular distribution mapping: High-resolution confocal microscopy can determine whether S1PR2 localizes to specific subcellular compartments within brain endothelial cells in response to stress.

• Quantitative analysis: Image analysis software can quantify S1PR2 immunopositivity in microvessels, correlating expression levels with parameters of vascular leakage in experimental models.

• Combined functional assays: Pairing antibody staining with in vivo vascular permeability assays using exogenous tracers (Evans blue, fluorescent dextrans) can directly correlate S1PR2 expression with functional barrier disruption.

This approach has revealed that S1PR2 plays a critical role in cerebrovascular permeability following ischemia/reperfusion injury, with S1PR2 immunopositivity specifically detected in ischemic microvessels of wild-type mice and in cerebrovascular endothelium of human brain autopsy samples.

What are common challenges when using FITC-conjugated S1PR2 antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with FITC-conjugated S1PR2 antibodies:

• High background fluorescence:

  • Cause: Insufficient blocking or tissue autofluorescence

  • Solution: Extend blocking time to 2 hours; try alternative blocking agents (5% normal serum); include 0.1-0.3% Triton X-100 for membrane permeabilization; use Sudan Black B (0.1%) treatment to reduce tissue autofluorescence

• Weak signal intensity:

  • Cause: Low S1PR2 expression, suboptimal fixation, or FITC photobleaching

  • Solution: Optimize fixation duration; use antigen retrieval methods; ensure proper storage of antibody away from light; consider signal amplification systems

• Non-specific binding:

  • Cause: Cross-reactivity with other S1P receptors

  • Solution: Validate antibody specificity using S1PR2 knockout tissues as negative controls; perform pre-absorption controls with the immunizing peptide

• Inconsistent staining patterns:

  • Cause: Variable S1PR2 expression under different conditions

  • Solution: Standardize experimental conditions; include positive controls from tissues known to express S1PR2 (e.g., cerebral microvessels after ischemia)

How can researchers validate the specificity of FITC-conjugated S1PR2 antibodies?

Validating specificity of FITC-conjugated S1PR2 antibodies requires a multi-faceted approach:

• Genetic controls: The most definitive validation method is testing antibodies on tissues from S1PR2 knockout (S1pr2-/-) mice, which should show complete absence of specific staining as demonstrated in previous studies.

• Blocking peptide competition: Pre-incubating the antibody with excess immunizing peptide (recombinant fragment within human S1PR2 aa 250 to C-terminus) should abolish specific staining.

• Western blot correlation: Parallel Western blot analysis of the same samples used for immunofluorescence can confirm the antibody detects a protein of the expected molecular weight for S1PR2.

• siRNA knockdown validation: In cell culture systems, comparing staining between S1PR2 siRNA-treated and control cells can verify antibody specificity.

• Multi-antibody concordance: Using multiple antibodies targeting different epitopes of S1PR2 should produce similar staining patterns in positive control tissues.

• Species cross-reactivity assessment: Testing on multiple species and comparing staining patterns can help confirm conservation of epitope recognition.

• Expression pattern correlation: The observed staining pattern should correlate with known tissue distribution of S1PR2, particularly in vascular endothelium under inflammatory or ischemic conditions.

How can FITC-conjugated S1PR2 antibodies be used to study the role of S1PR2 in stroke and neurovascular injury?

FITC-conjugated S1PR2 antibodies enable sophisticated investigation of S1PR2's role in stroke and neurovascular injury through several methodological approaches:

• Temporal expression profiling: Systematic immunofluorescence analysis at multiple time points following experimental stroke (tMCAO model) can reveal the dynamics of S1PR2 upregulation in cerebral microvessels.

• Spatial mapping of vulnerable regions: Comparing S1PR2 expression patterns across different brain regions can identify vascular beds particularly susceptible to S1PR2-mediated permeability.

• Cell-specific co-localization studies: Combining S1PR2 immunofluorescence with markers for endothelial cells (CD31), pericytes (PDGFRβ), and astrocytes (GFAP) can define the cellular distribution of S1PR2 within the neurovascular unit.

• Correlation with permeability markers: Pairing S1PR2 immunofluorescence with indicators of blood-brain barrier breakdown (extravasated IgG, fibrinogen, or albumin) can establish direct spatial relationships between S1PR2 expression and barrier dysfunction.

• Intervention response assessment: Comparing S1PR2 expression patterns between vehicle-treated and JTE013 (S1PR2 antagonist)-treated animals can reveal how pharmacological inhibition affects receptor expression and localization.

Research has demonstrated that S1PR2 plays a critical role in cerebrovascular permeability following ischemia-reperfusion injury, with genetic deletion or pharmacological inhibition of S1PR2 significantly reducing cerebral edema, hemorrhagic transformation, and neuronal death while improving neurological outcomes.

What methodologies can be used to study S1PR2-G13 protein interactions using FITC-conjugated antibodies?

Investigating S1PR2-G13 protein interactions using FITC-conjugated antibodies requires sophisticated methodological approaches:

• Proximity Ligation Assay (PLA): This technique can visualize S1PR2-Gα13 interactions in situ with subcellular resolution:

  • Incubate tissue sections with primary antibodies against S1PR2 (FITC-conjugated) and Gα13

  • Apply secondary antibodies conjugated with oligonucleotides

  • Ligation and amplification steps generate fluorescent spots only where proteins are in close proximity (<40 nm)

  • Quantify interaction signals in different subcellular compartments

• FRET (Förster Resonance Energy Transfer): For studying dynamic interactions:

  • Use FITC-conjugated S1PR2 antibodies as donor fluorophores

  • Label Gα13 with acceptor fluorophores in compatible spectral range

  • Measure energy transfer efficiency to determine molecular proximity

  • Analyze changes in FRET efficiency following S1P stimulation or drug treatments

• Co-immunoprecipitation with in situ detection:

  • Perform co-IP of S1PR2 and Gα13 from cell or tissue lysates

  • Validate interaction using Western blot

  • Correlate biochemical findings with immunofluorescence patterns in matching samples

  • Determine how specific mutations (e.g., in ICL2) affect both binding and localization

• Super-resolution microscopy:

  • Apply techniques like STORM or PALM to visualize S1PR2-Gα13 complexes beyond the diffraction limit

  • Quantify nanoscale distribution patterns in relation to cellular structures

  • Assess changes in complex formation following agonist stimulation

These approaches can help validate and extend findings from cryo-EM studies showing that S1PR2-Gα13 complex formation involves critical interactions between S1PR2's intracellular loop 2 and the α5 helix of Gα13.

How should researchers quantify and interpret S1PR2 expression patterns from immunofluorescence data?

Robust quantification and interpretation of S1PR2 immunofluorescence data requires systematic methodological approaches:

• Standardized image acquisition parameters:

  • Maintain consistent exposure settings, gain, and offset across experimental groups

  • Capture images at multiple magnifications (10x for overview, 40-63x for detailed analysis)

  • Collect z-stack series to account for tissue depth variations

• Quantification methods for vessel-associated S1PR2:

  • Co-localization analysis: Calculate Pearson's or Mander's coefficients to measure S1PR2/CD31 overlap

  • Intensity measurements: Measure mean fluorescence intensity within vessel-defined regions of interest

  • Distribution profiling: Generate intensity line profiles across vessel cross-sections

  • Density quantification: Calculate percentage of S1PR2-positive vessels relative to total vessel count

• Statistical approaches:

  • Compare multiple brain regions (cortex, striatum, hippocampus) within each animal

  • Analyze multiple sections per animal (minimum 3-5 sections at standard anatomical coordinates)

  • Include sufficient biological replicates (n=5-8 animals per experimental group)

  • Apply appropriate statistical tests (paired t-test for bilateral comparisons, ANOVA for multiple groups)

• Controls for interpretation:

  • Include S1PR2 knockout tissue to establish threshold for non-specific background

  • Compare against baseline expression in non-manipulated animals

  • Validate antibody specificity through Western blot correlation

• Contextual interpretation:

  • Correlate S1PR2 expression with functional outcomes (neurological scores, edema measurements)

  • Compare expression patterns with other markers of vascular dysfunction

This approach has revealed that S1PR2 immunopositivity is specifically detected in ischemic microvessels of wild-type mice but absent in non-ischemic tissue, demonstrating the context-dependent upregulation of this receptor.

What comparative analyses can be performed between S1PR2 expression and functional vascular permeability?

Researchers can employ several methodological approaches to correlate S1PR2 expression with functional vascular permeability:

Table 1. Methodological Approaches for Correlating S1PR2 Expression with Vascular Permeability

Analytical workflow for correlation studies:

• Perform permeability assessment using one or more techniques above

• Process adjacent tissue sections for S1PR2 immunofluorescence

• Generate region-matched quantitative data for both parameters

• Apply correlation analysis (Pearson's or Spearman's) to identify statistical relationships

• Generate scatterplots with regression analysis to visualize relationships

This integrated approach has revealed strong correlations between S1PR2 expression in cerebral microvessels and increased vascular permeability following ischemic injury, with both parameters significantly reduced in mice lacking S1PR2 or treated with S1PR2 antagonists.

How can S1PR2 antibody-based assays be used to evaluate potential therapeutic compounds?

S1PR2 antibody-based assays provide valuable methodological approaches for evaluating therapeutic compounds targeting this receptor:

• Receptor occupancy assays:

  • Treat cells/tissues with candidate compounds at various concentrations

  • Apply FITC-conjugated S1PR2 antibodies that compete for the same binding site

  • Measure reduction in fluorescence intensity to determine compound binding

  • Generate dose-response curves to calculate binding affinities

• Receptor internalization studies:

  • Monitor S1PR2 surface expression versus intracellular localization following drug treatment

  • Quantify changes in membrane/cytoplasmic distribution ratios

  • Track receptor trafficking through endosomal compartments

  • Correlate internalization patterns with functional outcomes

• Conformational change detection:

  • Utilize conformation-sensitive antibodies that recognize active/inactive states

  • Monitor changes in epitope accessibility following compound binding

  • Apply FRET-based approaches to detect structural rearrangements

• Target engagement in disease models:

  • Evaluate S1PR2 expression before and after therapeutic intervention

  • Correlate changes in receptor expression with functional improvements

  • Apply in models where S1PR2 plays established roles (stroke, hearing impairment)

• Combination with functional readouts:

  • Pair antibody-based detection with functional assays such as:

    • Endothelial cell monolayer resistance (ECIS)

    • Matrix metalloproteinase (MMP) activity assays

    • Cell migration assays

    • TUNEL assays for apoptosis detection

Research has demonstrated that inhibition of S1PR2 with antagonists like JTE013 significantly reduces cerebral edema and hemorrhagic transformation in experimental stroke models, with corresponding reductions in MMP-9 activity and improvements in neurological outcomes, providing a framework for therapeutic development.

What are the methodological considerations for studying S1PR2 in human pathological samples using immunofluorescence?

Studying S1PR2 in human pathological samples requires specific methodological considerations:

• Sample acquisition and processing:

  • Post-mortem interval significantly impacts immunoreactivity—prioritize samples with <24h PMI

  • Fixation protocols must be standardized (4% PFA is preferred, avoid long-term formalin storage)

  • Antigen retrieval methods may be necessary (citrate buffer, pH 6.0 heat-mediated retrieval)

  • Section thickness optimization (10-30μm) balances signal intensity with tissue penetration

• Antibody validation for human tissue:

  • Confirm antibody reactivity with human S1PR2 epitopes (sequence alignment analysis)

  • Perform peptide competition assays to verify specificity

  • Include positive control tissues with known S1PR2 expression

  • Compare staining patterns with multiple antibodies targeting different epitopes

• Autofluorescence management:

  • Human brain tissue contains significant lipofuscin that autofluoresces in the FITC spectrum

  • Treatment with Sudan Black B (0.1-0.3%) or TrueBlack reduces autofluorescence

  • Spectral imaging with unmixing algorithms can separate specific signal from autofluorescence

• Comparative analysis strategies:

  • Match cases and controls for age, sex, and relevant comorbidities

  • Standardize anatomical regions for comparison across samples

  • Implement blinded quantification by multiple observers

  • Correlate findings with clinical data and other molecular markers

• Multi-label approaches:

  • Combine S1PR2 with cell-type markers (CD31 for endothelium, GFAP for astrocytes)

  • Include markers of pathological processes (hypoxia, inflammation, apoptosis)

  • Apply sequential staining protocols for multiple antibodies from the same species

Previous studies have successfully detected S1PR2 immunopositivity in the cerebrovascular endothelium of human brain autopsy samples, providing translational relevance to findings from experimental models.

How can FITC-conjugated S1PR2 antibodies be integrated with single-cell analysis technologies?

Integration of FITC-conjugated S1PR2 antibodies with single-cell technologies enables powerful new research approaches:

• Flow cytometry-based applications:

  • Single-cell sorting: Isolate S1PR2+ cell populations from complex tissues based on fluorescence intensity

  • Multi-parameter phenotyping: Combine with other surface markers to identify specific vascular subpopulations expressing S1PR2

  • Phospho-flow analysis: Correlate S1PR2 expression with activation of downstream signaling pathways at single-cell resolution

  • Protocols: Gentle tissue dissociation with collagenase/dispase preserves surface epitopes; add Fc block to reduce non-specific binding

• Single-cell transcriptomics integration:

  • FACS-seq approach: Sort S1PR2+ and S1PR2- populations for comparative scRNA-seq analysis

  • CITE-seq method: Combine FITC-conjugated S1PR2 antibodies with oligo-tagged antibodies to measure both surface protein and gene expression simultaneously

  • Spatial transcriptomics: Correlate S1PR2 protein localization with transcriptional profiles in tissue context

• Advanced microscopy applications:

  • IMC (Imaging Mass Cytometry): Pair metal-tagged S1PR2 antibodies with dozens of other markers for deep phenotyping

  • Expansion microscopy: Apply physical tissue expansion techniques to resolve subcellular S1PR2 distribution beyond diffraction limits

  • Live-cell imaging: Monitor real-time changes in S1PR2 distribution following stimulation or drug treatment

• Methodological considerations:

  • Optimize antibody concentration for specific single-cell applications (typically higher dilution than for standard immunofluorescence)

  • Validate with appropriate isotype controls

  • Include compensation controls when combining with other fluorophores

These approaches can reveal heterogeneity in S1PR2 expression across endothelial cell subpopulations and correlation with specific cellular states during pathological conditions.

What role does S1PR2 play in modulating immune cell function and how can this be studied with antibody-based approaches?

S1PR2 plays multifaceted roles in immune cell function that can be investigated using antibody-based methodological approaches:

• Expression profiling across immune cell subsets:

  • Flow cytometry panels: Combine FITC-conjugated S1PR2 antibodies with lineage markers for T cells, B cells, macrophages, and dendritic cells

  • Tissue-resident vs. circulating populations: Compare expression between blood, lymphoid tissues, and sites of inflammation

  • Activation-dependent regulation: Monitor changes in S1PR2 expression following stimulation with cytokines, TLR ligands, or disease-relevant conditions

• Functional impact assessment:

  • Migration assays: Correlate S1PR2 expression with chemotactic responses in transwell systems

  • Adhesion studies: Evaluate interactions between S1PR2+ immune cells and vascular endothelium under flow conditions

  • Cytokine production: Sort cells based on S1PR2 expression and assess functional differences in cytokine production profiles

• Signaling pathway analysis:

  • Phosphoprotein detection: Combine S1PR2 staining with phospho-specific antibodies to evaluate downstream signaling

  • G protein coupling preferences: Assess co-localization with different G protein subunits in immune cells versus vascular cells

  • Cross-talk with immune receptors: Investigate interactions between S1PR2 and pattern recognition receptors or cytokine receptors

• In vivo tracking and manipulation:

  • Adoptive transfer experiments: Track differentially labeled S1PR2+ and S1PR2- cells to determine homing and functional differences

  • Conditional knockout models: Compare immune responses between wild-type and cell-specific S1PR2 knockout animals

  • Intravital microscopy: Monitor S1PR2+ immune cell behavior at vascular interfaces during inflammation

These approaches can help elucidate the role of S1PR2 in various immunological contexts, building on findings that S1PR2 signaling affects vascular responses to inflammatory stimuli and mediates interactions with immune cells during neurovascular injury.