vsig10 Antibody

What is VSIG10 and what is its significance in immunological research?

VSIG10 (V-Set and Immunoglobulin Domain Containing 10) is a type I transmembrane protein belonging to the immunoglobulin superfamily. It contains four Ig-like C2-type domains in its extracellular domain and is structurally related to the B7 family of immune regulatory proteins . Unlike other VSIG family members that have IgV domains, VSIG10 and VSIG10L (UniProt Accession # Q8N0Z9 and Q86VR7) lack this domain but possess IgC2 domains . VSIG10 is expressed in various tissues, including dendritic cells and intestinal epithelium, and has been implicated in negative regulation of CD4+ T cell activation . This makes it a potential target for immunotherapy research, particularly in the context of cancer treatment where immune checkpoint blockade is an emerging therapeutic approach.

How does VSIG10 compare structurally and functionally with other members of the VSIG family?

VSIG10 differs structurally from other VSIG family members in several key ways:

VSIG10 shares 95%, 69%, and 71% amino acid identity with chimpanzee, mouse, and rat VSIG10, respectively . Its unique structural characteristics suggest distinctive functional properties compared to other VSIG family members, though they all appear to play roles in immunoregulation .

What are the key applications for VSIG10 antibodies in basic research?

VSIG10 antibodies have several important applications in basic research:

• Immunohistochemistry (IHC): Detection of VSIG10 expression in various tissues, including brain and thyroid cancer samples .

• Western Blotting (WB): Analysis of VSIG10 protein expression levels in cell and tissue lysates .

• Immunocytochemistry (ICC)/Immunofluorescence (IF): Localization of VSIG10 within cells, as demonstrated in RT-4 human urinary bladder transitional cell papilloma cell line .

• Flow cytometry: Analysis of VSIG10 expression on cell surfaces.

• Protein-protein interaction studies: Investigation of VSIG10's binding partners and signaling pathways.

• Functional assays: Examining VSIG10's role in T cell activation and cytokine production.

These applications allow researchers to investigate VSIG10's distribution, regulation, and function in various biological contexts, particularly in immune regulation and cancer research .

What are the optimal conditions for using anti-VSIG10 antibodies in immunohistochemistry?

When using anti-VSIG10 antibodies for immunohistochemistry, researchers should consider the following optimal conditions:

• Antibody dilution: The recommended dilution range is typically 1:30-1:150 for IHC applications as suggested for certain VSIG10 polyclonal antibodies .

• Antigen retrieval: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is often effective for VSIG10 detection.

• Detection system: For paraffin-embedded sections, using polymer detection systems with HRP/DAB is recommended for optimal visualization.

• Positive controls: Human brain and thyroid cancer tissues have been verified as positive controls for VSIG10 antibodies .

• Blocking: Use appropriate blocking solutions (3-5% normal serum from the same species as the secondary antibody) to reduce background.

• Incubation conditions: Primary antibody incubation is typically performed at 4°C overnight or at room temperature for 1-3 hours, depending on the specific antibody.

• Counterstaining: Light hematoxylin counterstaining can be used to visualize tissue architecture without obscuring antibody staining.

For immunofluorescence applications, fluorophore-conjugated secondary antibodies such as NorthernLights™ 557-conjugated Anti-Mouse IgG have been successfully used with VSIG10 primary antibodies .

How should researchers validate the specificity of VSIG10 antibodies?

Validating VSIG10 antibody specificity is crucial for ensuring reliable research results. A comprehensive validation approach includes:

• Western blot analysis: Confirm that the antibody detects a protein of the expected molecular weight (~540 amino acids for full-length human VSIG10) .

• Knockout/knockdown controls: Use VSIG10 knockout cell lines like the VSIG10 Knockout HeLa cell line as negative controls to confirm antibody specificity.

• Peptide competition assay: Pre-incubate the antibody with the immunizing peptide (such as the synthetic peptide located between aa107-156 of human VSIG10 used for some antibodies) before applying to samples. Specific staining should be blocked.

• Cross-reactivity testing: Test the antibody against related proteins (other VSIG family members) to ensure it doesn't cross-react.

• Multiple antibody approach: Use different antibodies targeting distinct epitopes of VSIG10 and compare staining patterns.

• Immunoprecipitation followed by mass spectrometry: This can confirm that the antibody is truly pulling down VSIG10.

• Recombinant protein controls: Use recombinant VSIG10 proteins as positive controls.

• Species reactivity testing: Confirm predicted reactivity across species. For example, some VSIG10 antibodies show high sequence homology and cross-reactivity with human, gorilla, mouse, rat (100%), gibbon, marmoset, dog, bovine, rabbit, horse, pig (92%), and galago (84%) .

What approaches can be used to optimize VSIG10 antibody performance in Western blotting?

To optimize VSIG10 antibody performance in Western blotting, researchers should consider:

• Sample preparation:

  • Use appropriate lysis buffers containing protease inhibitors to prevent degradation

  • Include phosphatase inhibitors if phosphorylation status is important

  • Optimize protein loading (typically 20-50 μg of total protein)

• Gel electrophoresis conditions:

  • Use 8-10% gels for better resolution of VSIG10 (~60 kDa)

  • Include positive control samples (cells known to express VSIG10)

• Transfer parameters:

  • For large proteins like VSIG10, use lower current and longer transfer times

  • Consider using PVDF membranes rather than nitrocellulose for better protein retention

• Blocking conditions:

  • Test different blocking agents (5% non-fat dry milk, 5% BSA, commercial blocking buffers)

  • Block for 1-2 hours at room temperature or overnight at 4°C

• Antibody dilution and incubation:

  • Follow manufacturer recommendations for primary antibody dilution

  • Optimize incubation time and temperature (typically overnight at 4°C)

  • Use gentle agitation during incubation

• Washing steps:

  • Perform multiple (4-5) washes with TBS-T (0.1% Tween-20)

  • Extend washing time (5-10 minutes per wash) to reduce background

• Detection system:

  • Use high-sensitivity ECL substrate for low abundance proteins

  • Consider using fluorescent secondary antibodies for quantitative analysis

• Troubleshooting common issues:

  • For weak signals: increase antibody concentration, extend incubation time, or use signal enhancers

  • For high background: increase washing steps, reduce antibody concentration, or try different blocking agents

How can researchers distinguish between VSIG10 isoforms using specific antibodies?

Distinguishing between VSIG10 isoforms requires careful antibody selection and experimental design:

• Epitope-specific antibodies: Select antibodies targeting regions that differ between isoforms. For example, antibodies targeting the amino acid region 107-156 of human VSIG10 may detect specific isoforms depending on whether this region is conserved across isoforms.

• Western blot analysis with high-resolution gels: Use gradient gels (4-15%) to better separate closely related isoforms that may differ only slightly in molecular weight.

• 2D gel electrophoresis: Combine isoelectric focusing with SDS-PAGE to separate isoforms based on both charge and size differences.

• Isoform-specific primers for validation: Use RT-PCR with isoform-specific primers to confirm the presence of specific VSIG10 isoform mRNAs in your samples, which can be correlated with antibody detection.

• Mass spectrometry analysis: After immunoprecipitation with anti-VSIG10 antibodies, use mass spectrometry to identify peptides unique to specific isoforms.

• Recombinant isoform standards: Express and purify each VSIG10 isoform as recombinant proteins to serve as standards for comparing antibody reactivity and specificity.

• Knockout/knockin models: Use CRISPR-Cas9 to generate cells expressing only specific VSIG10 isoforms as validation tools.

• Post-translational modification-specific antibodies: If isoforms differ in their post-translational modifications, use antibodies that specifically recognize these modifications.

This multi-faceted approach enables precise identification of VSIG10 isoforms in complex biological samples.

What are the most effective strategies for detecting VSIG10 in different subcellular compartments?

Detecting VSIG10 in different subcellular compartments requires specialized techniques:

• Subcellular fractionation followed by Western blotting:

  • Separate nuclear, cytoplasmic, membrane, and organelle fractions

  • Use compartment-specific markers (e.g., Na⁺/K⁺-ATPase for plasma membrane, GAPDH for cytosol, histone H3 for nucleus) as controls

  • Probe each fraction with anti-VSIG10 antibodies

• High-resolution confocal microscopy:

  • Use immunofluorescence with VSIG10 antibodies

  • Co-stain with organelle markers (e.g., WGA for Golgi, MitoTracker for mitochondria)

  • Perform z-stack imaging to create 3D reconstructions of VSIG10 distribution

  • As observed in validated samples, VSIG10 has been detected in the cytoplasm of RT-4 human urinary bladder transitional cell papilloma cell line

• Super-resolution microscopy techniques:

  • STORM or PALM imaging for nanoscale localization

  • Structured illumination microscopy (SIM) for improved resolution

• Proximity ligation assay (PLA):

  • Detect interactions between VSIG10 and compartment-specific proteins

  • This provides spatial information about protein-protein interactions

• Electron microscopy with immunogold labeling:

  • Highest resolution for precise subcellular localization

  • Use gold-conjugated secondary antibodies to detect VSIG10 primary antibodies

• Live-cell imaging with fluorescently tagged VSIG10:

  • Create fluorescent protein fusions to track dynamic localization

  • Validate findings with antibody staining of fixed cells

• Flow cytometry with selective permeabilization:

  • Use differential detergents to selectively permeabilize cellular compartments

  • Detect internal vs. surface pools of VSIG10

Since VSIG10 is known to be an integral component of the membrane , these techniques can help determine its precise localization and trafficking between cellular compartments.

How do post-translational modifications affect VSIG10 antibody recognition, and how can this be addressed experimentally?

Post-translational modifications (PTMs) can significantly affect VSIG10 antibody recognition, which presents both challenges and opportunities for researchers:

• Impact of common PTMs on antibody recognition:

  • Glycosylation: Can mask epitopes or create steric hindrance

  • Phosphorylation: May alter protein conformation or create new epitopes

  • Ubiquitination: Can interfere with antibody binding or alter protein migration

  • Proteolytic processing: May remove epitopes or create new ones

• Experimental approaches to address PTM-related issues:

  a) Enzymatic treatment:

  • Use PNGase F to remove N-linked glycosylation

  • Use phosphatases to remove phosphate groups

  • Compare antibody recognition before and after treatment

  b) PTM-specific antibodies:

  • Develop or obtain antibodies that specifically recognize or are unaffected by particular PTMs

  • Use pairs of antibodies recognizing modified and unmodified forms

  c) Mass spectrometry analysis:

  • Map PTMs on VSIG10 in your experimental system

  • Correlate PTM profiles with antibody recognition patterns

  d) Site-directed mutagenesis:

  • Create VSIG10 mutants lacking specific modification sites

  • Test antibody recognition of these mutants

  e) Multiple epitope targeting:

  • Use antibodies targeting different epitopes (e.g., N-terminal, C-terminal, and internal domains)

  • The VSIG10 antibody targeting amino acids 107-156 may recognize different PTM states than antibodies targeting other regions

  f) Combined biochemical approaches:

  • 2D gel electrophoresis to separate VSIG10 based on charge changes from PTMs

  • Sequential immunoprecipitation with different antibodies to enrich specific modified forms

• Validation strategies:

  • Compare results from multiple antibodies recognizing different epitopes

  • Use recombinant VSIG10 with defined PTMs as controls

  • Include samples treated with PTM inhibitors as references

Understanding the effect of PTMs on VSIG10 antibody recognition is crucial for accurate interpretation of experimental results, especially when studying VSIG10 function in different cellular contexts and disease states.

How can VSIG10 antibodies be utilized to study the role of VSIG10 in cancer immunotherapy?

VSIG10 antibodies are valuable tools for investigating VSIG10's potential role in cancer immunotherapy:

• Mechanistic studies of T cell regulation:

  • Use blocking antibodies to disrupt VSIG10 interactions and assess effects on T cell activation

  • Monitor changes in cytokine production (particularly IL-17 secretion, which VSIG10 has been shown to suppress)

  • Analyze T cell proliferation and effector function in the presence of anti-VSIG10 antibodies

• Tumor microenvironment analysis:

  • Use immunohistochemistry to map VSIG10 expression in tumor tissues and tumor-infiltrating immune cells

  • Perform multiplex staining to correlate VSIG10 expression with immune cell markers and activation states

  • Compare expression patterns between responsive and non-responsive patients

• Functional screening of therapeutic antibodies:

  • Test panels of VSIG10 antibodies for their ability to block immunosuppressive functions

  • Assess antibody-dependent cellular cytotoxicity (ADCC) potential against VSIG10-expressing cells

  • Patent information indicates monoclonal antibodies targeting VSIG10's soluble ectodomain may have therapeutic potential in cancer

• Combination therapy studies:

  • Evaluate VSIG10 blockade in combination with established checkpoint inhibitors (anti-PD-1, anti-CTLA-4)

  • Assess synergistic effects on immune activation and tumor control

• Biomarker development:

  • Determine if VSIG10 expression levels correlate with response to immunotherapy

  • Develop VSIG10 antibody-based assays for patient stratification

• Genetic studies with validation by antibodies:

  • Use VSIG10 knockout cell lines alongside antibody-based detection to validate findings

  • Compare VSIG10 expression and function across different cancer types using antibody-based techniques

These approaches can help establish whether VSIG10 represents a viable target for cancer immunotherapy, similar to other VSIG family members like TIGIT that have shown promise in this field .

What are the experimental considerations when using VSIG10 antibodies to investigate immune checkpoint mechanisms?

When investigating VSIG10 as a potential immune checkpoint using antibodies, researchers should consider:

• Antibody format selection:

  • Full IgG vs. Fab or F(ab')₂ fragments (to eliminate Fc receptor interactions)

  • Consider using antibodies with defined isotypes to control for Fc-mediated effects

  • Monoclonal vs. polyclonal antibodies (monoclonals offer greater specificity and reproducibility)

• Functional assay design:

  • T cell activation assays: measure proliferation, cytokine production, and surface activation markers

  • Co-culture systems with antigen-presenting cells expressing VSIG10

  • Three-dimensional culture systems to better mimic tissue microenvironments

  • In vivo models using humanized mice to test antibody effects on human immune cells

• Epitope targeting strategy:

  • Target different domains of VSIG10 to identify functional epitopes

  • Compare antibodies targeting the IgC2 domains vs. other regions

  • Determine if targeting the amino acid region 107-156 affects functional outcomes

• Controls and validation:

  • Include isotype control antibodies

  • Use VSIG10 knockout systems as negative controls

  • Compare effects with established checkpoint inhibitors

  • Validate findings across multiple immune cell types and donors

• Receptor-ligand interaction analysis:

  • Use surface plasmon resonance or biolayer interferometry to measure binding kinetics

  • Perform competition assays to identify antibodies that block natural ligand binding

  • Use co-immunoprecipitation to identify binding partners in relevant cell types

• Translational considerations:

  • Test antibodies on primary human samples from diverse donors

  • Evaluate species cross-reactivity for preclinical model development

  • Consider antibody humanization if therapeutic development is planned

• Technical optimizations:

  • Determine optimal antibody concentrations that avoid non-specific effects

  • Establish appropriate timing for antibody addition in functional assays

  • Consider Fc receptor blocking in assays using full IgG antibodies

These considerations will help ensure robust and reproducible results when investigating VSIG10's potential role as an immune checkpoint molecule.

How do VSIG10 expression patterns correlate with disease progression, and what methodological approaches using antibodies can best elucidate these relationships?

Investigating correlations between VSIG10 expression and disease progression requires systematic antibody-based approaches:

• Tissue microarray (TMA) analysis:

  • Use validated VSIG10 antibodies on TMAs containing samples from different disease stages

  • Perform quantitative image analysis to score expression levels

  • VSIG10 has been detected in adenocarcinoma samples , and systematic analysis could reveal stage-specific patterns

• Multiplex immunohistochemistry/immunofluorescence:

  • Combine VSIG10 antibodies with markers for immune cells, proliferation, and tissue-specific markers

  • Use spectral unmixing to analyze co-expression patterns

  • Correlate with patient outcome data to identify prognostic signatures

• Single-cell analysis techniques:

  • Flow cytometry with VSIG10 antibodies to analyze expression at the single-cell level

  • Mass cytometry (CyTOF) for high-dimensional analysis of VSIG10 in relation to multiple markers

  • Single-cell sorting followed by functional assays to determine the role of VSIG10+ cells

• Longitudinal sampling approaches:

  • Analyze VSIG10 expression in sequential biopsies or liquid biopsies

  • Correlate changes in expression with treatment response or disease progression

  • Develop standardized protocols for consistent antibody-based detection over time

• RNA-protein correlation studies:

  • Combine RNA-seq or qPCR data with antibody-based protein detection

  • Identify discrepancies between transcription and translation that may have functional significance

  • Validate findings with multiple antibodies targeting different VSIG10 epitopes

• Clinicopathological correlation:

  • Create antibody-based scoring systems for VSIG10 expression

  • Perform multivariate analysis to determine independent prognostic value

  • Use digital pathology platforms for standardized quantification

• Functional validation in disease models:

  • Antibody-mediated depletion or blocking studies in relevant disease models

  • Use VSIG10 knockout cell lines for comparison with antibody-based findings

  • Evaluate effects of modulating VSIG10 on disease progression markers

This comprehensive approach can establish whether VSIG10 expression serves as a biomarker for disease progression or therapeutic response, particularly in cancers where other VSIG family members have shown prognostic significance .

What are the common pitfalls when working with VSIG10 antibodies, and how can researchers overcome them?

Researchers working with VSIG10 antibodies may encounter several challenges:

• Non-specific binding:

  • Problem: Background staining or multiple bands in Western blots

  • Solution: Optimize blocking conditions (try different blocking agents like BSA, milk, or commercial blockers); increase washing steps; titrate antibody concentration; use more stringent washing buffers

• Epitope masking:

  • Problem: Inability to detect VSIG10 despite known expression

  • Solution: Try different antigen retrieval methods (heat-induced vs. enzymatic); use antibodies targeting different epitopes (e.g., antibodies targeting region aa107-156 vs. those targeting other regions); optimize fixation protocols for immunohistochemistry applications

• Variable detection across applications:

  • Problem: Antibody works well in Western blot but poorly in IHC or vice versa

  • Solution: Select application-validated antibodies (e.g., some VSIG10 antibodies are specifically validated for IHC , while others are validated for Western blot); consider using different antibodies for different applications

• Cross-reactivity with other VSIG family members:

  • Problem: False positive signals due to detection of related proteins

  • Solution: Validate specificity using knockout models ; perform peptide competition assays; select antibodies targeting unique regions of VSIG10 not conserved in other family members

• Batch-to-batch variability:

  • Problem: Inconsistent results with different antibody lots

  • Solution: Request lot-specific validation data from manufacturers; maintain reference samples for comparison; consider monoclonal antibodies which typically show less lot-to-batch variation than polyclonals

• Low sensitivity for detecting endogenous VSIG10:

  • Problem: Weak or undetectable signals when analyzing endogenous expression

  • Solution: Use signal amplification methods (e.g., tyramide signal amplification for IHC); concentrate samples for Western blot; use more sensitive detection systems; consider immunoprecipitation to enrich VSIG10 before detection

• Interference from post-translational modifications:

  • Problem: Variable detection depending on PTM status

  • Solution: Use enzymatic treatments to remove specific modifications; select antibodies that are not affected by common PTMs; use antibodies specifically recognizing modified forms if studying PTMs

• Subcellular localization challenges:

  • Problem: Difficulty detecting VSIG10 in specific cellular compartments

  • Solution: Use appropriate fixation and permeabilization protocols optimized for membrane proteins; perform subcellular fractionation before Western blotting; use confocal microscopy with z-stack imaging for better localization

• Species cross-reactivity issues:

  • Problem: Unpredicted or absence of cross-reactivity with animal models

  • Solution: Check sequence homology before antibody selection; validate species cross-reactivity experimentally; some VSIG10 antibodies show high homology across species (human, gorilla, mouse, rat) , making them suitable for comparative studies

How can researchers address conflicting results between different VSIG10 antibodies?

When confronted with conflicting results between different VSIG10 antibodies, researchers should implement a systematic approach to identify the source of discrepancies:

• Epitope mapping analysis:

  • Determine the exact binding sites of each antibody

  • Compare results from antibodies targeting different domains of VSIG10

  • Consider whether certain epitopes might be masked in specific experimental contexts

• Multi-antibody validation approach:

  • Use at least three independent antibodies targeting different epitopes

  • Compare results across multiple applications (WB, IHC, IF, etc.)

  • Establish consensus findings versus antibody-specific artifacts

• Correlation with nucleic acid-based detection:

  • Verify VSIG10 expression using qPCR or RNA-seq

  • Use in situ hybridization to validate tissue localization patterns

  • Compare protein levels detected by different antibodies with mRNA levels

• Knockout/knockdown controls:

  • Test all antibodies on VSIG10 knockout cell lines

  • Implement siRNA or CRISPR knockdown approaches to create graduated levels of expression

  • Determine which antibodies accurately reflect expression changes

• Recombinant protein standards:

  • Test antibody recognition of recombinant VSIG10 with known concentration

  • Compare detection sensitivity and linearity across antibodies

  • Use tagged recombinant proteins to confirm antibody specificity

• Technical optimization comparison:

  • Systematically test each antibody across a range of conditions

  • Determine whether discrepancies are due to technical factors rather than antibody specificity

  • Document optimal conditions for each antibody

• Cross-validation with orthogonal techniques:

  • Compare antibody-based results with mass spectrometry data

  • Use proximity ligation assays to confirm protein interactions

  • Employ functional assays to correlate expression with activity

• Manufacturer consultation:

  • Contact antibody providers for technical support

  • Inquire about known limitations or caveats

  • Request detailed validation data beyond what's in the datasheet

• Protocol standardization:

  • Use identical protocols when comparing antibodies

  • Control for variables like fixation, antigen retrieval, and detection methods

  • Document all experimental conditions in detail

• Collaborative validation:

  • Send samples to independent laboratories for blinded analysis

  • Compare results from different research groups using the same antibodies

  • Establish consensus findings across multiple research environments

This systematic approach can help determine which antibodies provide the most reliable results and identify the sources of discrepancies, ultimately leading to more robust research findings.

What are the key methodological considerations for optimizing VSIG10 detection in challenging sample types?

Detecting VSIG10 in challenging sample types requires specialized approaches:

• Formalin-fixed, paraffin-embedded (FFPE) tissues:

  • Challenge: Epitope masking due to fixation

  • Optimization: Test multiple antigen retrieval methods (citrate buffer pH 6.0, EDTA buffer pH 9.0, enzymatic retrieval); extend retrieval times; use higher antibody concentrations

  • Technique: Apply antibody dilutions in the range of 1:30-1:150 as recommended for certain VSIG10 antibodies in IHC applications

• Archival or degraded samples:

  • Challenge: Protein degradation affecting epitope integrity

  • Optimization: Use antibodies targeting stable epitopes; employ signal amplification systems; combine multiple antibodies targeting different regions

  • Validation: Include positive control tissues with known VSIG10 expression like brain and thyroid cancer samples

• Low-expression samples:

  • Challenge: Signal below detection threshold

  • Optimization: Employ tyramide signal amplification; use high-sensitivity detection systems; increase incubation times; concentrate samples before analysis

  • Analysis: Use digital image analysis with algorithms designed for low-signal detection

• Lipid-rich tissues:

  • Challenge: High background and poor antibody penetration

  • Optimization: Use detergent-containing buffers; extend washing steps; try different fixatives; perform delipidation steps before antibody incubation

  • Controls: Include isotype controls to distinguish specific staining from lipid-associated background

• Highly autofluorescent tissues:

  • Challenge: Signal masking by autofluorescence

  • Optimization: Use autofluorescence quenching treatments; select detection fluorophores with spectral properties distinct from autofluorescence; employ spectral unmixing during image acquisition

  • Alternative: Use chromogenic detection methods instead of fluorescence

• Frozen tissue sections:

  • Challenge: Morphological preservation and antibody compatibility

  • Optimization: Test different fixation protocols (acetone, methanol, PFA); optimize section thickness; use hydrophobic barriers to prevent antibody runoff

  • Technique: Apply appropriate blocking to reduce background staining

• Blood and circulating cell samples:

  • Challenge: Low VSIG10 expression on specific cell subsets

  • Optimization: Use flow cytometry with sensitive fluorophores; employ bead-based signal amplification; optimize cell preparation to preserve membrane proteins

  • Analysis: Gate on specific cell populations to analyze VSIG10 in rare subsets

• Primary cell cultures:

  • Challenge: Expression changes during culture

  • Optimization: Minimize culture time before analysis; use conditions that maintain in vivo phenotype; compare with directly analyzed tissue

  • Validation: Correlate antibody staining with mRNA expression in the same samples

• Multiplex detection systems:

  • Challenge: Antibody compatibility in multiplex panels

  • Optimization: Test antibody performance in singleplex before multiplex; carefully select compatible fluorophores; use sequential staining protocols if needed

  • Controls: Include single-stain controls for accurate compensation/unmixing

• Three-dimensional samples (organoids, thick sections):

  • Challenge: Antibody penetration issues

  • Optimization: Extend incubation times; use smaller antibody fragments; employ detergents to improve penetration; optimize clearing protocols

  • Imaging: Use confocal or light-sheet microscopy with appropriate depth corrections

These optimizations can significantly improve VSIG10 detection in challenging sample types, enabling more comprehensive analysis across diverse experimental contexts.