CLEC10A Antibody, HRP conjugated

What is CLEC10A and why is it important in immunological research?

CLEC10A, also known as macrophage galactose/N-acetyl-galactosamine (GalNAc) specific lectin (MGL), CD301, DC-ASGPR, or HML, is a 40 kDa type II transmembrane glycoprotein belonging to the C-type lectin family. Its significance in immunological research stems from its expression on immature myeloid dendritic cells and alternatively activated (tolerogenic) macrophages. CLEC10A selectively binds and internalizes terminal nonsialylated alpha- or beta-linked GalNAc moieties on O-linked carbohydrates, including the Tn carcinoma antigen. This receptor plays a crucial role in inhibiting effector T cell activation by binding to carbohydrate determinants on CD45 isoforms expressed by T, NK, and B cells . Recent research has also demonstrated CLEC10A's importance in skin homeostasis, particularly against house dust mite (HDM)-induced dermatitis .

How do I optimize CLEC10A antibody dilutions for different applications?

Optimal dilutions for CLEC10A antibodies vary significantly depending on the application, sample type, and detection method. For HRP-conjugated antibodies, preliminary titration experiments are essential. Begin with manufacturer-recommended dilutions and test a range (typically 1:500 to 1:5000) for Western blots and 1:50 to 1:500 for immunohistochemistry. When optimizing for flow cytometry, start with 5-10 μg/mL and adjust based on signal-to-noise ratio. For all applications, include appropriate positive controls (such as tolerogenic macrophages or dendritic cells) and negative controls. Optimal dilutions should be determined by each laboratory for each specific application, as indicated in manufacturer protocols .

What are the recommended fixation and permeabilization protocols when using CLEC10A antibodies for immunohistochemistry?

For optimal results with CLEC10A antibodies in immunohistochemistry, tissue samples should be fixed in 10% neutral-buffered formalin for 24-48 hours, followed by paraffin embedding. For antigen retrieval, heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) has shown good results. When working with cell preparations, 4% paraformaldehyde fixation for 15 minutes at room temperature is recommended. For intracellular staining, permeabilization with 0.1-0.5% Triton X-100 for 5-10 minutes is typically effective. These protocols have been successfully used in studies examining CLEC10A binding in formalin-fixed, paraffin-embedded normal and cancerous mammary tissues . As with all antibody applications, optimization for specific experimental conditions is recommended.

How can I distinguish between human CLEC10A and its mouse homologs (MGL1/MGL2) in cross-species research?

Distinguishing between human CLEC10A and its mouse homologs requires careful antibody selection and experimental design due to significant structural and functional differences. While humans possess a single gene for CLEC10A/MGL, mice have two closely related genes: MGL1 and MGL2. Within the carbohydrate recognition domain (CRD), human CLEC10A/MGL shares 64-70% amino acid sequence identity with mouse MGL1, mouse MGL2, and rat MGL .

For cross-species research, use antibodies validated for specificity, such as clone 744812, which has been confirmed in direct ELISAs to recognize human CLEC10A/CD301 without cross-reactivity to mouse MGL1 . For functional studies, note that human CLEC10A and mouse MGL2 share similar ligand preferences for terminal GalNAc moieties, while mouse MGL1 differs in its binding profile . When analyzing experimental data, these functional differences must be considered, particularly when extrapolating mouse data to human systems.

What methodological approaches can be used to investigate CLEC10A's role in dermatitis models?

Investigating CLEC10A's role in dermatitis models requires a multi-faceted approach combining genetic, immunological, and histological techniques:

• Genetic models: Utilize CRISPR-Cas9 gene editing to repair or introduce mutations in the Clec10a gene, as demonstrated in the NC/Nga mouse model where repairing a stop-gain mutation ameliorated house dust mite (HDM)-induced dermatitis .

• Knockout models: Employ Clec10a-/- mice on C57BL/6J background to assess exacerbated HDM-induced dermatitis compared to wild-type controls .

• Ex vivo analysis: Isolate skin macrophages to evaluate CLEC10A's inhibitory effect on HDM-induced Toll-like receptor 4 (TLR4)-mediated inflammatory cytokine production through its inhibitory immunoreceptor tyrosine activating motif .

• Ligand identification and application: Identify mucin-like molecules in HDM as potential ligands for mouse Clec10a and human Asgr1, and assess their therapeutic potential by topical application to ameliorate TLR4 ligand-induced dermatitis .

• Immunohistochemistry: Use HRP-conjugated CLEC10A antibodies to detect and quantify receptor expression in skin tissue sections, correlating with dermatitis severity.

What are the current limitations of using recombinant CLEC10A domains versus antibodies in glycan detection?

Recombinant CLEC10A domains and CLEC10A antibodies present distinct advantages and limitations for glycan detection:

Recombinant CLEC10A domains:

• Advantages: Detect physiological ligands with native binding specificity; can identify tumor-associated glycans that potentially interact with tumor-associated macrophages (TAMs); and provide information about functional interactions between glycans and their receptors .

• Limitations: Require careful validation with truncated CRD controls; may have lower binding affinity compared to polyvalent constructs; and detection depends on c-myc tag antibodies, adding complexity to the detection system .

CLEC10A antibodies:

• Advantages: Directly detect the receptor rather than its ligands; available in multiple formats including HRP-conjugated versions for direct detection; and well-characterized specificity profiles .

• Limitations: Do not provide information about receptor-ligand interactions; may cross-react with structurally similar proteins without proper validation; and antibody binding may be affected by glycosylation of the receptor itself.

For comprehensive glycan studies, combining both approaches provides complementary data: recombinant domains identify potential ligands while antibodies confirm receptor expression and localization.

How do different conjugation methods affect CLEC10A antibody performance?

Different conjugation methods can significantly impact CLEC10A antibody performance across various applications:

The choice of conjugation should be based on the specific research application. For HRP-conjugated CLEC10A antibodies, optimal applications include chromogenic immunohistochemistry and Western blotting, where direct enzymatic detection provides advantages in terms of protocol simplicity and sensitivity .

What controls should be included when validating CLEC10A antibody specificity?

A comprehensive validation strategy for CLEC10A antibodies should include multiple controls:

• Positive tissue controls: Use tissues with known CLEC10A expression, such as tissues containing alternatively activated (M2) macrophages or immature myeloid dendritic cells .

• Negative tissue controls: Include tissues known to lack CLEC10A expression or tissues from CLEC10A knockout animals.

• Peptide competition/blocking: Pre-incubate the antibody with excess purified CLEC10A protein or peptide to confirm binding specificity.

• Cross-reactivity assessment: Test against closely related proteins such as asialoglycoprotein receptor 1 (Asgr1) or mouse MGL1/MGL2 to ensure specificity .

• Isotype controls: Include relevant isotype-matched control antibodies with the same conjugation to assess non-specific binding.

• Truncated domain controls: For functional studies, include truncated CRD domain constructs as negative controls, as demonstrated in protein domain histochemistry approaches .

• Multiple antibody comparison: When possible, validate findings using antibodies targeting different epitopes of CLEC10A.

Proper validation ensures that experimental results can be confidently attributed to specific CLEC10A detection rather than non-specific binding or cross-reactivity.

How can CLEC10A antibodies be utilized to study the relationship between glycosylation patterns and cancer progression?

CLEC10A antibodies offer valuable tools for investigating the relationship between aberrant glycosylation and cancer progression:

• Tumor glycan profiling: HRP-conjugated CLEC10A antibodies can be used to detect Tn and STn antigens (truncated O-glycans) in tumor tissue microarrays, correlating their expression with clinical outcomes and metastatic potential .

• Monitoring glycosylation changes: Track alterations in glycan patterns during cancer progression, treatment response, and hormone therapy. Research has shown that CLEC10A-positive glycan structures are induced by 4-hydroxy-tamoxifen and associated with alterations in glycan synthesis in breast cancer cell lines .

• Tumor-associated macrophage (TAM) interactions: Investigate how TAMs expressing CLEC10A interact with tumor cells displaying altered glycosylation, potentially influencing tumor microenvironment and progression .

• Therapeutic target identification: Identify patients who might benefit from therapies targeting TAM-tumor glycan interactions by screening for specific glycan profiles recognized by CLEC10A .

• Comparative studies: Use antibodies against both CLEC10A and its ligands to comprehensively assess glycosylation changes across different cancer types, stages, and in response to therapies.

This research approach has particular relevance in breast cancer, where significant differences in CLEC10A binding between normal mammary glands and tumor tissues have been observed .

What are the optimal methods for studying CLEC10A-mediated signaling in immune regulation?

Studying CLEC10A-mediated signaling in immune regulation requires a combination of cellular, biochemical, and molecular approaches:

• Co-immunoprecipitation studies: Use HRP-conjugated or unconjugated CLEC10A antibodies to precipitate receptor complexes from tolerogenic macrophages or dendritic cells, followed by mass spectrometry analysis to identify signaling partners.

• Phosphorylation analysis: Examine phosphorylation of the inhibitory immunoreceptor tyrosine activating motif in CLEC10A's cytoplasmic portion following receptor engagement, focusing on its impact on TLR4-mediated inflammatory signaling .

• Functional assays: Measure cytokine production, specifically inflammatory cytokines, in the presence of CLEC10A-activating ligands and/or blocking antibodies to assess the receptor's regulatory function.

• Receptor clustering analysis: Use fluorescently-labeled antibodies and advanced microscopy to visualize CLEC10A clustering following ligand binding, correlating with signaling outcomes.

• Glycan-receptor binding kinetics: Employ surface plasmon resonance with recombinant CLEC10A extracellular domains to determine binding affinities for various glycan structures, informing structure-function relationships .

• In vivo models: Utilize wild-type and Clec10a-/- mice to examine immune responses to inflammatory challenges, focusing on macrophage and dendritic cell activation states .

These approaches collectively provide insights into how CLEC10A modulates immune regulation through its interaction with glycan structures on immune cells and pathogens.

How might CLEC10A antibodies contribute to understanding the role of this receptor in viral infection pathways?

CLEC10A antibodies can significantly advance our understanding of viral infection mechanisms through several research strategies:

• Viral glycoprotein interactions: Use HRP-conjugated CLEC10A antibodies in binding assays to identify and characterize viral glycoproteins that interact with this receptor. Research has shown that CLEC10A/MGL binds the GP envelope glycoprotein on Marburg and Ebola viruses, enhancing viral entry and infectivity .

• Infection pathway visualization: Apply immunofluorescence techniques with CLEC10A antibodies to visualize receptor-mediated viral entry, trafficking, and processing within macrophages and dendritic cells.

• Blocking studies: Employ antibodies as blocking agents to assess the functional importance of CLEC10A in viral entry and subsequent immune responses.

• Glycan profile alterations: Monitor changes in CLEC10A ligand expression during viral infection using recombinant receptor domains alongside antibodies.

• Comparative analysis: Compare CLEC10A's role in different viral infections to identify common mechanisms of glycan-mediated immune evasion.

This research direction is particularly relevant for enveloped viruses that display terminal GalNAc structures recognized by CLEC10A, potentially revealing novel therapeutic targets for antiviral interventions.

What methodologies can be employed to investigate the potential therapeutic applications of targeting CLEC10A in inflammatory skin conditions?

Investigating CLEC10A's therapeutic potential in inflammatory skin conditions can be approached through multiple methodologies:

• Targeted delivery systems: Develop antibody-drug conjugates or nanoparticles decorated with CLEC10A ligands to deliver anti-inflammatory compounds specifically to receptor-expressing macrophages in inflamed skin.

• Topical ligand application: Building on findings that mucin-like molecules from house dust mites can serve as CLEC10A ligands, develop and evaluate topical formulations containing these ligands to ameliorate TLR4-induced dermatitis, as demonstrated in mouse models .

• Receptor engagement monitoring: Use HRP-conjugated antibodies in skin biopsies to assess CLEC10A expression levels before and after treatment interventions, correlating with clinical improvement.

• Human-mouse comparative studies: Investigate both CLEC10A in mice and Asgr1 (its functional homolog) in humans to ensure translational relevance of findings .

• Ex vivo skin models: Employ organotypic skin cultures from normal and inflammatory skin disease patients to test CLEC10A-targeting compounds in a controlled environment.

• Genetic rescue experiments: Utilize CRISPR-Cas9 technology to repair CLEC10A mutations in animal models, assessing impact on disease phenotype as demonstrated with the stop-gain mutation in NC/Nga mice .

These approaches offer promising avenues for developing novel therapeutics that modulate macrophage function through CLEC10A engagement, potentially addressing unmet needs in inflammatory dermatological conditions.