VSIG4 Antibody, Biotin conjugated

What is VSIG4 and why is it significant in immunological research?

VSIG4, also known as CRIg or Z39IG, is a 45 kDa type I transmembrane protein belonging to the B7 family within the immunoglobulin superfamily. It has significant importance in immunological research due to its specific expression pattern and multiple immunoregulatory functions. VSIG4 functions as a phagocytic receptor and serves as a strong negative regulator of T-cell proliferation and IL-2 production. Additionally, it acts as a potent inhibitor of the alternative complement pathway convertases . These characteristics make VSIG4 a valuable target for studying macrophage function, complement regulation, and T-cell responses in both normal physiology and disease states.

The gene encoding VSIG4 is located on the X chromosome, and the human VSIG4 cDNA encodes 399 amino acids including a 19 aa signal sequence, a 264 aa extracellular domain containing V-type and C2-type Ig domains, a 21 aa transmembrane segment, and a 95 aa cytoplasmic domain . This molecular structure enables VSIG4 to participate in diverse cellular interactions, particularly in immune regulation and homeostasis.

What is the tissue distribution pattern of VSIG4 expression?

VSIG4 exhibits a highly specific expression pattern limited to tissue-resident macrophages, making it an excellent marker for studying this macrophage subpopulation. The protein is specifically expressed on macrophages in:

• Thymic medulla

• Peritoneum

• Alveoli

• Synovia

• Adipose tissue

• Heart

• Liver Kupffer cells

• Placental Hofbauer cells

• Atherosclerotic foam cells

Notably, VSIG4 is absent on infiltrating macrophages, which provides a means to distinguish between resident and recruited macrophage populations in various tissues . This distinct expression profile makes VSIG4 antibodies valuable tools for identifying and characterizing tissue-resident macrophages in both healthy and pathological contexts, particularly in diseases with significant macrophage involvement.

What are the known splice variants of VSIG4 and their functional implications?

Several splice isoforms of VSIG4 have been identified, with proteins of 321, 305, 272, 201, and 199 amino acids reported in the literature. These variants differ in their structural composition, with some lacking all or part of the cytoplasmic domain, the C2-type Ig domain, and/or the transmembrane domain . The existence of these multiple isoforms suggests potential diverse regulatory mechanisms and functional roles of VSIG4 in different cellular contexts.

When designing experiments with VSIG4 antibodies, researchers should consider which isoforms their antibody recognizes and whether this might affect interpretation of results. Particularly for studies involving transcriptional analysis or protein detection, accounting for these splice variants is essential for accurate data interpretation and experimental reproducibility.

What are the key characteristics of VSIG4 Antibody, Biotin conjugated?

VSIG4 Antibody, Biotin conjugated is typically available as a polyclonal antibody derived from rabbit hosts, designed to recognize human VSIG4. The biotinylation provides enhanced sensitivity and flexibility in detection methods. Key specifications include:

The antibody has been tested and confirmed to work in multiple applications, with human samples showing consistent and reliable results . The biotin conjugation enables versatile detection methods using streptavidin-based systems, enhancing sensitivity and providing flexibility in experimental design.

How should VSIG4 Antibody, Biotin conjugated be stored and handled to maintain optimal activity?

Proper storage and handling of VSIG4 Antibody, Biotin conjugated is critical for maintaining its activity and specificity. Upon receipt, the antibody should be stored at -20°C or -80°C to ensure long-term stability . Researchers should avoid repeated freeze-thaw cycles as these can degrade the antibody and reduce its effectiveness.

When working with the antibody:

• Aliquot the stock solution into smaller volumes to minimize freeze-thaw cycles

• Allow the antibody to equilibrate to room temperature before opening the vial

• Use sterile technique when handling the antibody

• Return unused portions to -20°C or -80°C immediately after use

• Prepare working dilutions fresh for each experiment

For reconstituted lyophilized antibodies, they are generally stable for approximately 12 months from the date of receipt when stored at -20 to -70°C. After reconstitution, they can be stored for 1 month at 2 to 8°C under sterile conditions, or for up to 6 months at -20 to -70°C under sterile conditions .

What detection systems work optimally with VSIG4 Antibody, Biotin conjugated?

Since the antibody is biotin-conjugated, streptavidin-based detection systems are optimal for visualization. The biotin-streptavidin system provides significant signal amplification due to the high affinity between biotin and streptavidin, enhancing detection sensitivity. Common detection approaches include:

• Streptavidin-HRP (horseradish peroxidase) systems for colorimetric detection in ELISA, WB, and IHC

• Streptavidin-fluorophore conjugates for fluorescence-based detection in IHC or flow cytometry

• Streptavidin-alkaline phosphatase for alternative colorimetric detection

For ELISA applications, a sandwich enzyme immunoassay technique is commonly employed. In this approach, an antibody specific for Human VSIG4 is pre-coated onto a microplate. Samples containing VSIG4 are added to the wells, and the immobilized antibody binds the VSIG4 present in the sample. After washing away unbound substances, the biotin-conjugated detection antibody is added, binding to the captured VSIG4. Following another wash, streptavidin-HRP conjugate is added. After incubation and washing, a substrate solution is added, producing a color that develops in proportion to the amount of VSIG4 bound in the initial step .

How can VSIG4 Antibody, Biotin conjugated be optimized for immunohistochemistry applications?

Optimizing VSIG4 Antibody, Biotin conjugated for immunohistochemistry requires careful attention to several parameters to achieve specific staining with minimal background. Based on research applications, the following protocol elements should be considered:

• Tissue Preparation and Antigen Retrieval:

  • For paraffin-embedded tissues, complete deparaffinization and rehydration are essential

  • Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or EDTA buffer (pH 9.0) is recommended

  • Antigen retrieval time should be optimized based on tissue type and fixation method

• Blocking and Antibody Dilution:

  • Block endogenous biotin using a commercial biotin blocking kit to prevent non-specific binding

  • Block endogenous peroxidase with 3% hydrogen peroxide for 10 minutes

  • Use 5-10% normal serum from the same species as the secondary antibody for blocking

  • Optimize antibody dilution through titration; a starting dilution of 1:200 has shown good results in human lung, placenta, and skeletal muscle tissues

• Incubation and Detection:

  • Incubate primary antibody overnight at 4°C for optimal binding

  • After washing, use streptavidin-HRP at an appropriate dilution

  • Develop with DAB substrate and counterstain with hematoxylin

  • Mount with appropriate mounting medium

Successful IHC staining has been demonstrated in various human tissues including lung, placenta, and skeletal muscle, typically showing staining patterns consistent with tissue-resident macrophage distribution . Researchers should include positive control tissues known to express VSIG4 (such as liver, lung, or placenta) and negative controls (primary antibody omitted) in each experiment.

What are the recommended protocols for utilizing VSIG4 Antibody, Biotin conjugated in ELISA?

For optimal ELISA performance with VSIG4 Antibody, Biotin conjugated, a sandwich ELISA approach is recommended. The following protocol provides methodological guidance:

• Plate Preparation:

  • Coat a high-binding 96-well microplate with capture antibody specific for VSIG4 (typically 1-2 μg/mL) in coating buffer

  • Incubate overnight at 4°C

  • Wash and block with blocking buffer containing 1-5% BSA or normal serum

• Sample Preparation:

  • Prepare standards using recombinant VSIG4 protein in a range appropriate for the expected sample concentrations

  • Dilute samples appropriately in Standard/Sample Diluent (R1)

  • Add 100 μL of standards and samples to appropriate wells and incubate for 2 hours at room temperature

• Detection:

  • Wash wells thoroughly (at least 3-5 times)

  • Add diluted VSIG4 Antibody, Biotin conjugated (diluted 1:100 in Biotin-Conjugate Antibody Diluent R2)

  • Incubate for 1 hour at room temperature

  • Wash wells thoroughly

  • Add Streptavidin-HRP (diluted 1:100 in Streptavidin-HRP Diluent R3)

  • Incubate for 30 minutes at room temperature

  • Wash wells thoroughly

  • Add substrate solution and incubate until appropriate color development

  • Add stop solution and read absorbance at 450 nm with correction at 570 nm

This sandwich ELISA method has been validated for detecting VSIG4 in human serum, plasma, cell culture supernatants, tissue homogenates, and other biological fluids . The method provides quantitative measurement of VSIG4 levels with high specificity and sensitivity.

How can researchers validate the specificity of VSIG4 Antibody, Biotin conjugated?

Validating antibody specificity is crucial for ensuring reliable experimental results. For VSIG4 Antibody, Biotin conjugated, several validation approaches are recommended:

• Positive and Negative Control Tissues:

  • Positive controls: Use tissues known to express VSIG4, such as liver (Kupffer cells), lung (alveolar macrophages), and placenta (Hofbauer cells)

  • Negative controls: Use tissues with minimal VSIG4 expression or include an experimental arm where the primary antibody is omitted

• Western Blot Validation:

  • Run recombinant VSIG4 protein alongside tissue lysates known to express VSIG4

  • The antibody should detect a band at approximately 44 kDa (predicted molecular weight of VSIG4)

  • Perform peptide competition assays using the immunizing peptide to confirm specificity

• siRNA Knockdown:

  • Use siRNA-mediated knockdown of VSIG4 in an appropriate cell type (such as macrophages)

  • Compare antibody staining between knockdown and control cells

  • A specific antibody will show reduced staining in knockdown cells

• Comparative Antibody Analysis:

  • Test multiple antibodies targeting different epitopes of VSIG4

  • Compare staining patterns across different tissues

  • Consistent staining patterns across antibodies suggest specificity

• Mass Spectrometry Validation:

  • Immunoprecipitate VSIG4 using the antibody

  • Analyze the precipitated proteins by mass spectrometry

  • Confirmation of VSIG4 in the precipitated fraction supports antibody specificity

Both antibody blockade of VSIG4 and siRNA-mediated knockdown approaches have been successfully used to study VSIG4 function in human macrophage biology, providing validation methods for antibody specificity while also yielding functional insights .

How does VSIG4 regulate macrophage function, and how can VSIG4 antibodies be used to study this process?

VSIG4 plays a critical role in regulating macrophage function through several mechanisms. It serves as a complement receptor that binds C3b and iC3b fragments, internalizes them to recycling endosomes, and is then recycled to the cell surface . This process contributes significantly to innate immunity through binding and phagocytosis of complement-opsonized pathogens.

Recent research has revealed that VSIG4 also mediates transcriptional inhibition of Nlrp3 and Il-1β in macrophages, suggesting a role in regulating inflammatory responses . Furthermore, VSIG4 has been shown to interact with MS4A6D to form a surface inhibitory signaling complex (SISC) that suppresses the expression of NLRP3 and IL-1β during inflammatory responses, activating the JAK2-STAT3-A20 signaling pathway .

Researchers can use VSIG4 antibodies to study these processes through several approaches:

• Macrophage Polarization Studies:

  • VSIG4 antibodies can be used to identify and isolate specific macrophage populations based on VSIG4 expression

  • Blockade of VSIG4 with antibodies has been shown to repolarize M2 macrophages toward a more inflammatory phenotype, suggesting therapeutic potential

• Signaling Pathway Analysis:

  • Coimmunoprecipitation experiments using VSIG4 antibodies can identify interaction partners such as MS4A6D

  • Western blot analysis of downstream signaling molecules (JAK2, STAT3, A20) can elucidate the signaling cascade activated by VSIG4

• Functional Assays:

  • Phagocytosis assays using VSIG4 antibodies to block function can reveal its role in pathogen clearance

  • Complement regulation assays can assess how VSIG4 inhibits the alternative complement pathway

  • T-cell proliferation assays can measure how VSIG4 regulates T-cell activation

These approaches have been instrumental in demonstrating that VSIG4 plays an essential role in negative regulation of macrophage-driven intracellular inflammation .

What is known about the role of VSIG4 in cancer immunotherapy, and how can researchers utilize VSIG4 antibodies in this context?

Recent research has revealed VSIG4 as a promising target for cancer immunotherapy. Antibodies targeting VSIG4 have been shown to repolarize tumor-associated macrophages (TAMs) from an immunosuppressive M2-like phenotype to a more pro-inflammatory phenotype, inducing an immune response that culminates in T cell activation .

In vitro, in vivo, and ex vivo assays have demonstrated that anti-VSIG4 antibodies induce pro-inflammatory cytokines in M-CSF plus IL-10-driven human monocyte-derived M2c macrophages. Across patient-derived tumor samples from multiple tumor types, anti-VSIG4 treatment resulted in the upregulation of cytokines associated with TAM repolarization and T cell activation, as well as chemokines involved in immune cell recruitment .

Importantly, VSIG4 blockade has shown efficacy in a syngeneic mouse model as monotherapy and enhances efficacy when combined with anti-PD-1 therapy. This effect is dependent on the systemic availability of CD8+ T cells, suggesting that VSIG4 represents a promising new target capable of triggering an anti-cancer response via multiple key immune mechanisms .

Researchers can utilize VSIG4 antibodies in cancer immunotherapy research through:

• Ex vivo tumor sample analysis:

  • Treating patient-derived tumor samples with anti-VSIG4 antibodies to assess changes in cytokine production and immune cell activation

  • Using flow cytometry with VSIG4 antibodies to characterize TAM populations before and after treatment

• In vivo modeling:

  • Using anti-VSIG4 antibodies alone or in combination with checkpoint inhibitors in syngeneic tumor models

  • Monitoring tumor growth, survival, and immune infiltration

• Mechanism of action studies:

  • Investigating how VSIG4 blockade affects macrophage phenotype and function

  • Analyzing downstream signaling events triggered by VSIG4 targeting

These approaches can help elucidate the full potential of VSIG4 as a target for cancer immunotherapy and identify patient populations most likely to benefit from this approach.

What methodological approaches are recommended for studying VSIG4-MS4A6D interactions?

VSIG4 has been shown to interact with MS4A6D to form a surface inhibitory signaling complex that suppresses expression of NLRP3 and IL-1β in macrophages . Studies of this interaction provide important insights into VSIG4's regulatory functions. Several methodological approaches are recommended for investigating this interaction:

• Yeast Split-Ubiquitin Screening System:

  • This approach has been successfully used to identify MS4A6D as an interaction partner of VSIG4

  • The method involves using mouse Vsig4 C-terminal ubiquitin fusion (pBT3-SUC-Vsig4) as bait against a cDNA library constructed from mouse peritoneal macrophages

• Coimmunoprecipitation (Co-IP):

  • Co-IP experiments have demonstrated that endogenous VSIG4 and MS4A6D interact in peritoneal macrophages

  • Binding VSIG4 with monoclonal antibodies (such as VG11 mAbs) appears to enhance VSIG4/MS4A6D interactions

  • This technique can also show that MS4A6D directly binds JAK2, with antibody treatment enhancing this interaction

• Immunofluorescence Double Staining:

  • This technique has been used to demonstrate that MS4A6D colocalizes with VSIG4 on macrophage membranes

  • The approach provides visual confirmation of the spatial relationship between these proteins

• Protein Truncation Assessments:

  • Transient overexpression experiments in RAW264.7 cells have confirmed that VSIG4 coimmunoprecipitates with MS4A6D

  • Further protein truncation assessments have shown that amino acid residues between 1 and 46 at the N-terminus of MS4A6D are crucial for mediating these interactions

• Functional Studies:

  • Following identification of the interaction, functional studies can assess how this interaction affects downstream signaling

  • Experimental approaches should examine how this interaction influences the JAK2-STAT3-A20 signaling pathway

These methodological approaches provide complementary data on the physical and functional interaction between VSIG4 and MS4A6D, offering insights into how VSIG4 regulates macrophage inflammatory responses.

What are common challenges when using VSIG4 Antibody, Biotin conjugated, and how can they be addressed?

When working with VSIG4 Antibody, Biotin conjugated, researchers may encounter several challenges. Here are common issues and recommended solutions:

• High Background in Immunohistochemistry:

  • Cause: Inadequate blocking, particularly of endogenous biotin, or non-specific binding

  • Solution: Implement a stringent biotin blocking step before primary antibody incubation; increase BSA or serum concentration in blocking buffer; optimize primary antibody dilution (try 1:300-1:500 instead of 1:200)

• Weak or Absent Staining:

  • Cause: Insufficient antigen retrieval, over-fixation, or degraded epitopes

  • Solution: Optimize antigen retrieval conditions (try different pH buffers and longer retrieval times); ensure tissue samples are properly fixed but not over-fixed; confirm antibody activity with positive control tissues

• Inconsistent ELISA Results:

  • Cause: Variability in antibody performance or matrix effects from complex samples

  • Solution: Standardize all reagents and incubation times; prepare fresh standards for each assay; consider matrix-matched calibration curves; run duplicate or triplicate measurements

• Non-specific Bands in Western Blot:

  • Cause: Cross-reactivity with other proteins or degradation products

  • Solution: Optimize blocking conditions and antibody dilution; reduce primary antibody concentration; include a peptide competition control; ensure samples are freshly prepared with protease inhibitors

• Variable Expression Across Samples:

  • Cause: Biological variability or sample handling differences

  • Solution: Standardize sample collection and processing; include housekeeping protein controls; consider larger sample sizes to account for biological variability

When troubleshooting, it's important to remember that VSIG4 expression is highly specific to tissue-resident macrophages . Tissues with few resident macrophages might show minimal staining even with optimal protocols. Always include appropriate positive controls (such as liver Kupffer cells) and negative controls in experiments to validate results.

How should researchers interpret conflicting data on VSIG4 function across different experimental models?

Conflicting data on VSIG4 function across different experimental models is not uncommon and requires careful interpretation. Several factors may contribute to these discrepancies:

• Species Differences:

  • Human and mouse VSIG4 share structural similarities but also differences. Human VSIG4 contains both V-type and C2-type Ig domains, while mouse VSIG4 lacks the C2-type domain

  • When comparing across species, consider these structural differences and their potential functional implications

• Isoform Expression:

  • Multiple splice isoforms of VSIG4 exist (321, 305, 272, 201, and 199 aa), which may have different functions

  • Determine which isoforms are present in your experimental model and whether the antibody recognizes all relevant isoforms

• Contextual Factors:

  • VSIG4 function may be context-dependent; while it may activate NF-κB and be pro-inflammatory in some cases, many of its activities are important in resolving rather than initiating inflammation

  • Consider the specific microenvironment and activation state of cells in each experimental system

• Methodological Variations:

  • Different methods of VSIG4 inhibition (genetic knockout, siRNA knockdown, or antibody blockade) may yield different results due to complete versus partial loss of function

  • Standardize methodologies when possible or acknowledge these differences in interpretation

• Integrated Analysis Approach:

  • When faced with conflicting data, perform multiple complementary experiments

  • Use both gain-of-function and loss-of-function approaches

  • Validate findings across different cell types and experimental systems

  • Consider in vivo studies to complement in vitro findings

Recent research has shown that both antibody blockade of VSIG4 and siRNA-mediated knockdown can be used to study VSIG4 function in human macrophage biology . By employing multiple approaches and carefully considering the experimental context, researchers can develop a more comprehensive understanding of VSIG4's diverse functions.

What are the latest advances in understanding VSIG4's role in macrophage repolarization?

Recent research has revealed significant insights into VSIG4's role in macrophage polarization, particularly in the context of tumor microenvironments. Key findings include:

• Macrophage Phenotype Regulation:

  • VSIG4 has been identified as a critical regulator that maintains macrophages in an M2-like, immunosuppressive state

  • Anti-VSIG4 antibodies have been shown to repolarize M2 macrophages toward a more pro-inflammatory phenotype capable of coordinating an anti-tumor immune response

• Cytokine Production Modulation:

  • Antibody blockade of VSIG4 induces pro-inflammatory cytokines in M-CSF plus IL-10-driven human monocyte-derived M2c macrophages

  • In patient-derived tumor samples from multiple tumor types, anti-VSIG4 treatment results in upregulation of cytokines associated with TAM repolarization and T cell activation

• Chemokine Production and Immune Recruitment:

  • VSIG4 blockade leads to increased production of chemokines involved in immune cell recruitment to the tumor microenvironment

  • This suggests a role for VSIG4 in controlling not only macrophage phenotype but also the broader immune landscape

• T Cell Activation Pathway:

  • The repolarization of macrophages following VSIG4 blockade culminates in enhanced T cell activation

  • This effect appears to be dependent on the systemic availability of CD8+ T cells, indicating a critical link between VSIG4, macrophage polarization, and adaptive immunity

These advances highlight VSIG4 as a promising target for immunotherapy approaches aimed at reprogramming the tumor microenvironment, particularly in combination with other immunomodulatory agents such as checkpoint inhibitors.

How can VSIG4 Antibody, Biotin conjugated be utilized in single-cell analysis techniques?

VSIG4 Antibody, Biotin conjugated offers valuable opportunities for single-cell analysis techniques, enabling researchers to investigate VSIG4 expression and function at unprecedented resolution. Methodological approaches include:

• Single-Cell Flow Cytometry:

  • The biotin conjugation allows for flexible detection using streptavidin-fluorophore conjugates

  • Multi-parameter flow cytometry can identify VSIG4+ macrophage subpopulations in complex tissues

  • Protocol recommendations:

    • Use a streptavidin-fluorophore with minimal spectral overlap with other markers

    • Include appropriate compensation controls

    • Consider using a viability dye to exclude dead cells

• Mass Cytometry (CyTOF):

  • Biotin-conjugated antibodies can be detected using metal-tagged streptavidin

  • This approach allows for simultaneous detection of dozens of markers on single cells

  • VSIG4 can be included in comprehensive immune phenotyping panels to characterize macrophage heterogeneity

• Single-Cell RNA-Seq Validation:

  • While single-cell RNA-seq provides transcriptomic profiles, protein validation is often necessary

  • VSIG4 antibodies can be used to sort cell populations prior to sequencing or validate findings at the protein level

  • CITE-seq (Cellular Indexing of Transcriptomes and Epitopes by Sequencing) can incorporate biotin-conjugated antibodies for simultaneous protein and RNA detection

• Imaging Mass Cytometry or Multiplex Immunofluorescence:

  • These techniques allow visualization of VSIG4+ cells in their spatial context

  • Biotin-conjugated antibodies provide flexibility in detection strategies

  • This approach is particularly valuable for understanding VSIG4+ macrophage distribution in relation to other immune cells in tissues

These single-cell approaches can provide crucial insights into the heterogeneity of VSIG4 expression among macrophages, its correlation with other markers of macrophage polarization, and its spatial distribution in tissues under normal and pathological conditions.

What is the potential of VSIG4 as a therapeutic target, and how can antibodies contribute to preclinical studies?

VSIG4 has emerged as a promising therapeutic target, particularly in cancer immunotherapy. Several lines of evidence support its potential:

• Macrophage Repolarization:

  • Anti-VSIG4 antibodies repolarize M2 macrophages and induce an immune response culminating in T cell activation

  • This repolarization effect shifts the tumor microenvironment from immunosuppressive to immunostimulatory

• Efficacy in Preclinical Models:

  • VSIG4 blockade has shown efficacy in syngeneic mouse models as monotherapy

  • It enhances efficacy when combined with anti-PD-1 therapy, suggesting potential for combination immunotherapy approaches

• CD8+ T Cell Dependence:

  • The anti-tumor effect of VSIG4 blockade is dependent on the systemic availability of CD8+ T cells

  • This indicates that VSIG4 targeting can engage both innate and adaptive immune responses

• Ex Vivo Human Tumor Responses:

  • Treatment of patient-derived tumor samples from multiple tumor types with anti-VSIG4 antibodies results in upregulation of pro-inflammatory cytokines and chemokines

  • This suggests potential clinical translatability

Researchers can utilize antibodies in preclinical studies through:

• Target Validation Studies:

  • Use antibodies to confirm VSIG4 expression in relevant tissues and disease models

  • Employ multiple antibody clones to ensure robust target validation

• Mechanism of Action Studies:

  • Investigate how anti-VSIG4 antibodies affect macrophage phenotype, cytokine production, and T cell activation

  • Examine effects on complement regulation and phagocytosis

• Therapeutic Efficacy Assessment:

  • Test anti-VSIG4 antibodies in various cancer models as monotherapy and in combination with other immunotherapies

  • Evaluate dose-response relationships and pharmacokinetic/pharmacodynamic parameters

• Biomarker Development:

  • Identify potential biomarkers that predict response to VSIG4-targeted therapy

  • Develop companion diagnostic approaches using VSIG4 antibodies

The evidence suggests that VSIG4 represents a promising new immunotherapeutic target capable of triggering an anti-cancer response via multiple key immune mechanisms . Continued preclinical investigation will be essential to fully characterize its therapeutic potential and identify optimal clinical applications.

What controls should be included when working with VSIG4 Antibody, Biotin conjugated?

Appropriate controls are essential for ensuring the reliability and specificity of results when working with VSIG4 Antibody, Biotin conjugated. The following controls should be considered:

• Positive Controls:

  • Tissues or cells known to express VSIG4, such as:

    • Liver tissue (Kupffer cells)

    • Lung tissue (alveolar macrophages)

    • Placental tissue (Hofbauer cells)

    • Human macrophage cell lines or primary macrophages

• Negative Controls:

  • Primary antibody omission: Perform the complete protocol without the primary antibody

  • Isotype controls: Use a biotin-conjugated rabbit IgG at the same concentration as the VSIG4 antibody

  • Tissues with minimal VSIG4 expression (e.g., skeletal muscle has shown minimal staining)

• Specificity Controls:

  • Peptide competition/blocking: Pre-incubate the antibody with recombinant VSIG4 protein before application

  • siRNA knockdown: Compare staining in cells where VSIG4 has been knocked down versus control cells

  • Knockout tissues (if available): Compare staining in VSIG4 knockout versus wild-type tissues

• Technical Controls:

  • Endogenous biotin blocking controls: Essential in tissues with high endogenous biotin

  • Endogenous peroxidase blocking controls: Important for HRP-based detection systems

  • Serial dilution controls: To determine optimal antibody concentration

• Quantification Controls:

  • Standard curves with recombinant protein (for ELISA)

  • Loading controls (for Western blot)

  • Normalization controls (for quantitative analysis)

By incorporating these controls, researchers can validate the specificity of their VSIG4 antibody staining and ensure that observed patterns truly reflect VSIG4 expression rather than technical artifacts or non-specific binding.

What quantification methods are recommended for analyzing VSIG4 expression in different experimental contexts?

Accurate quantification of VSIG4 expression is essential for comparative analysis across different experimental conditions. Recommended quantification methods vary depending on the experimental approach:

• Immunohistochemistry Quantification:

  • Scoring Systems:

    • Percentage of positive cells (0-100%)

    • Staining intensity (0: negative, 1+: weak, 2+: moderate, 3+: strong)

    • H-score (combines percentage and intensity: 0-300)

  • Digital Image Analysis:

    • Use specialized software for unbiased quantification

    • Measure parameters such as positive pixel count, positive cell count, and staining intensity

    • Normalize to total tissue area or cell count

  • Recommendations:

    • Analyze multiple fields per sample (at least 5-10)

    • Blind scoring by multiple observers when possible

    • Include reference standards for consistent scoring

• ELISA Quantification:

  • Use a standard curve of recombinant VSIG4 protein for absolute quantification

  • Ensure samples fall within the linear range of the standard curve

  • Run samples in duplicate or triplicate

  • Convert absorbance values to concentration using four-parameter logistic regression

  • Report results as ng/mL or pg/mL of sample

• Western Blot Quantification:

  • Use densitometry software to quantify band intensity

  • Normalize to loading controls (β-actin, GAPDH, or total protein)

  • Include a standard curve of recombinant protein if absolute quantification is needed

  • Present results as relative expression compared to control conditions

• Flow Cytometry Quantification:

  • Measure median fluorescence intensity (MFI) for VSIG4 staining

  • Calculate percentage of VSIG4+ cells

  • Use markers of median equivalent soluble fluorophore (MESF) for standardization across experiments

  • Consider relative expression to isotype control or fluorescence minus one (FMO) controls

• RT-qPCR Quantification:

  • Use the 2^(-ΔΔCT) method for relative quantification

  • Normalize to appropriate housekeeping genes

  • Include melt curve analysis to confirm specificity

  • Consider absolute quantification using standard curves if comparing across multiple experiments

These quantification methods provide complementary data on VSIG4 expression at the protein and mRNA levels, allowing for comprehensive analysis across different experimental contexts.