CLEC4E Antibody

What is CLEC4E and why is it important for immunological research?

CLEC4E (C-type lectin domain family 4 member E), also known as Mincle or CLECSF9, is an approximately 30 kDa type 2 transmembrane C-type lectin that functions as an activating innate immune receptor . The human CLEC4E protein consists of a 19 amino acid cytoplasmic domain, a 21 amino acid transmembrane segment, and a 179 amino acid extracellular domain containing the C-type lectin domain . This receptor is primarily expressed on monocytes, macrophages, and immature dendritic cells, making it critical for pathogen recognition and inflammatory responses . CLEC4E is important for immunological research because it binds to mycobacterial glycolipids, including TDM (cord factor) and its synthetic analog TDB, as well as to endogenous danger signals like SAP130 released from necrotic cells . This dual ability to recognize both pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) positions CLEC4E as a key player in both infection and sterile inflammation research.

How do CLEC4E antibodies differ in their application capacities?

CLEC4E antibodies come in various formats with different application potentials:

Different antibody formats serve specific experimental needs . Monoclonal antibodies provide high specificity for defined epitopes, making them excellent for applications requiring consistent batch-to-batch reproducibility . Conjugated antibodies eliminate the need for secondary detection reagents, simplifying experimental workflows and reducing background in multicolor immunofluorescence or flow cytometry . The choice between these formats should be guided by the specific experimental requirements, target localization (membrane-bound vs. intracellular), and detection method sensitivity needed .

What are the critical controls needed when using CLEC4E antibodies?

When using CLEC4E antibodies, several critical controls should be implemented to ensure experimental validity:

• Positive control samples: Cells known to express CLEC4E (monocytes, macrophages) or CLEC4E-transfected cell lines should be included . The data from R&D Systems shows successful detection of CLEC4E in transfected HEK293 cells compared to cells transfected with irrelevant protein .

• Negative control samples: Cells known not to express CLEC4E or CLEC4E-knockout cells serve as essential negative controls .

• Isotype controls: Include the appropriate isotype control antibody (e.g., mouse IgG2b kappa for the B-7 clone) to assess non-specific binding .

• Secondary antibody controls: When using unconjugated primary antibodies, include controls with secondary antibody alone to evaluate background staining .

• Blocking peptide controls: Using the specific peptide that the antibody was raised against can confirm specificity through signal abolishment .

Implementing these controls helps distinguish true CLEC4E detection from artifacts and ensures reliable interpretation of experimental results across different applications.

How can CLEC4E antibodies be optimized for dual-color immunofluorescence with other C-type lectin receptors?

Optimizing CLEC4E antibodies for dual-color immunofluorescence with other C-type lectin receptors requires careful consideration of antibody combinations and protocol adjustments:

• Antibody species selection: Choose primary antibodies raised in different host species (e.g., mouse anti-CLEC4E and rabbit anti-CLEC4D) to allow simultaneous detection without cross-reactivity . If antibodies from the same species are unavoidable, consider directly conjugated antibodies or sequential staining protocols.

• Fluorophore selection: Select fluorophores with minimal spectral overlap. For instance, combining CLEC4E Alexa Fluor 700-conjugated antibody (FAB8995N) with a FITC-conjugated antibody against another receptor provides good spectral separation .

• Blocking strategy: Implement a robust blocking protocol using both serum from the species of secondary antibodies and Fc receptor blockers, as CLEC4E is expressed on Fc receptor-positive cells .

• Co-localization analysis: When studying CLEC4E's association with other receptors like CLEC4D/MCL, quantitative co-localization analysis can reveal functional complexes. Research has shown that CLEC4E associates with CLEC4D/MCL and gamma chain signaling subunits of Fc receptors, mediated by an Arg residue in the CLEC4E transmembrane segment .

• Fixation optimization: Test different fixation methods, as some epitopes may be sensitive to particular fixatives. Paraformaldehyde (2-4%) is generally suitable for CLEC4E detection, but methanol may better preserve certain epitopes .

Using these approaches enables reliable simultaneous detection of CLEC4E and other C-type lectin receptors, facilitating research on their cooperative roles in immune response regulation.

What methodological approaches can differentiate between CLEC4E activity in homeostatic versus inflammatory conditions?

Distinguishing CLEC4E activity between homeostatic and inflammatory conditions requires sophisticated methodological approaches:

• Temporal expression analysis: Monitor CLEC4E expression kinetics using flow cytometry with anti-CLEC4E antibodies at multiple time points after inflammatory stimuli . This reveals the dynamic regulation pattern specific to inflammatory conditions versus steady-state expression.

• Phosphorylation-specific detection: Develop experimental protocols that detect the phosphorylation state of downstream signaling molecules (Syk, CARD9, NF-κB) following CLEC4E engagement. This can be achieved using phospho-specific antibodies in western blot or flow cytometry analyses, revealing active signaling versus mere receptor presence .

• Ligand-induced internalization assays: Track receptor internalization after ligand binding using fluorescently-labeled anti-CLEC4E antibodies and confocal microscopy or flow cytometry. CLEC4E internalization rates differ significantly between homeostatic surveillance and active inflammatory response .

• Transcriptional profiling: Couple anti-CLEC4E immunoprecipitation with RNA-seq analysis of associated transcription factors to identify differential transcriptional programs between homeostatic and inflammatory states .

• In situ proximity ligation assay: This technique can visualize CLEC4E interactions with different co-receptors (CLEC4D/MCL) or signaling molecules that may differ between homeostatic and inflammatory conditions .

These methodological approaches provide multidimensional data on CLEC4E activity states, enabling researchers to distinguish receptor presence from functional activation in different physiological contexts.

How can CLEC4E antibodies be utilized to investigate cross-talk with other pattern recognition receptor pathways?

Investigating CLEC4E cross-talk with other pattern recognition receptor (PRR) pathways requires strategic antibody applications:

• Co-immunoprecipitation studies: Utilize anti-CLEC4E antibodies like the agarose-conjugated sc-390806 AC for pull-down experiments to identify protein complexes formed with other PRRs or their signaling components under different stimulation conditions . This reveals physical associations that may mediate cross-talk.

• Phospho-flow cytometry: Combine surface staining using anti-CLEC4E antibodies with intracellular phospho-specific antibodies against signaling molecules shared between CLEC4E and other PRR pathways (e.g., NF-κB, MAP kinases). This approach quantifies pathway activation at the single-cell level following selective or combined receptor stimulation .

• Receptor clustering analysis: Apply super-resolution microscopy with fluorescently-labeled anti-CLEC4E antibodies to visualize receptor clustering and co-localization with other PRRs like TLRs or NLRs after different ligand exposures .

• Sequential immunoprecipitation: Use anti-CLEC4E antibodies for primary immunoprecipitation followed by immunoblotting with antibodies against other PRRs or signaling molecules to identify specific interaction partners and their activation states .

• Chromatin immunoprecipitation (ChIP) analysis: Investigate transcriptional regulation downstream of CLEC4E signaling by analyzing promoter regions of inflammatory genes that might be co-regulated by multiple PRR pathways .

These approaches enable mapping of the complex signaling networks where CLEC4E participates, revealing synergistic or antagonistic relationships with other innate immune receptors that collectively shape immune responses.

Why might CLEC4E antibodies show inconsistent staining patterns in different immune cell types?

Inconsistent staining patterns of CLEC4E across immune cell types can stem from several factors:

• Variable expression levels: CLEC4E expression differs significantly between monocytes, macrophages, and dendritic cells, with expression being particularly high in monocytes and certain macrophage subsets. This natural variation can appear as inconsistent staining if not properly contextualized .

• Isoform diversity: Multiple alternatively spliced isoforms of CLEC4E exist, encoded by a gene located in the natural killer gene complex region on human chromosome 12. These isoforms may have different epitope accessibility or subcellular localization, leading to variable antibody binding patterns .

• Activation-dependent expression: CLEC4E expression is highly regulated by inflammatory stimuli, with significant upregulation following exposure to certain PAMPs and DAMPs. Cells at different activation states will display different staining intensities .

• Technical considerations:

  • Fixation effects: Different immune cell types may respond differently to the same fixation protocol, affecting epitope preservation

  • Permeabilization sensitivity: Membrane integrity varies between immune cell types, leading to differential permeabilization and antibody access

  • Fc receptor interference: Variable Fc receptor expression across immune cells can cause non-specific binding or blocking issues

• Solution approaches:

  • Optimize fixation and permeabilization for each cell type

  • Include blocking steps specific for Fc receptors

  • Validate with multiple antibody clones recognizing different epitopes

  • Consider flow cytometry with quantitative beads to standardize detection sensitivity

Understanding these factors allows researchers to develop cell type-specific protocols that yield consistent and reliable CLEC4E detection across diverse immune populations.

How can researchers resolve conflicting data between different detection methods for CLEC4E?

When encountering conflicting data between different CLEC4E detection methods, a systematic troubleshooting approach is essential:

• Comprehensive epitope analysis: Different antibodies may recognize distinct epitopes that are differentially accessible depending on the detection method. Compare the epitope regions targeted by each antibody and assess how sample preparation might affect their accessibility .

• Sample preparation comparison:

  Method	Protein State	Common Issues	Resolution Strategy
  Western Blot	Denatured	Size discrepancy	Use reducing/non-reducing conditions to assess multimeric status
  Flow Cytometry	Native, surface	Low signal	Optimize fixation, use amplification systems
  Immunohistochemistry	Fixed, crosslinked	Epitope masking	Test antigen retrieval methods
  ELISA	Variable	Interference	Validate with blocking peptides

• Cross-validation strategy: Implement a multi-method approach where at least three independent techniques (e.g., western blot, flow cytometry, and immunofluorescence) are used with the same antibody, and results are compared to establish consensus detection .

• Antibody validation using genetic controls: Test antibodies against CLEC4E-knockout samples or CLEC4E-overexpressing systems as definitive controls. The data from R&D Systems shows clear differentiation between CLEC4E-transfected and control-transfected cells, providing a validation standard .

• Technical optimization: For each technique, optimize critical parameters:

  • Western blot: Test different lysis buffers to ensure complete solubilization

  • Flow cytometry: Compare surface versus intracellular staining protocols

  • Immunofluorescence: Evaluate different fixation and permeabilization methods

By implementing this systematic approach, researchers can reconcile seemingly conflicting data and establish a reliable detection protocol for CLEC4E across different experimental systems.

What strategies can overcome weak CLEC4E antibody signals in tissue immunohistochemistry?

Enhancing weak CLEC4E antibody signals in tissue immunohistochemistry requires multiple optimization strategies:

• Antigen retrieval optimization: Test multiple antigen retrieval methods, as CLEC4E epitopes may be particularly sensitive to fixation artifacts:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Tris-EDTA buffer (pH 9.0) for improved retrieval of certain epitopes

  • Enzymatic retrieval using proteinase K for membrane proteins like CLEC4E

• Signal amplification systems:

  • Implement tyramide signal amplification (TSA) to enhance chromogenic or fluorescent signals

  • Use biotin-streptavidin amplification systems with CLEC4E antibodies

  • Consider polymer-based detection systems that provide multi-enzyme attachment per antibody

• Antibody concentration and incubation optimization:

  • Extend primary antibody incubation to overnight at 4°C to improve binding kinetics

  • Test higher antibody concentrations while monitoring background signals

  • Implement multiple sequential antibody applications with gentle washing between steps

• Background reduction strategies:

  • Include tissue-specific protein blocking (e.g., casein for lymphoid tissues)

  • Add detergents like Tween-20 at 0.05-0.1% to reduce non-specific hydrophobic interactions

  • Pre-absorb antibodies against tissue homogenates from negative control samples

• Technical considerations:

  • Use freshly cut tissue sections to minimize epitope loss from oxidation

  • Consider multiplex immunohistochemistry with other myeloid markers as internal controls

  • Implement automated staining platforms for consistent reagent application and washing

These comprehensive strategies can significantly enhance CLEC4E detection sensitivity in tissue sections while maintaining specificity, enabling accurate assessment of expression patterns in health and disease contexts.

How do researchers address species differences when studying CLEC4E with antibodies?

Addressing species differences in CLEC4E research requires careful antibody selection and experimental design:

• Sequence homology considerations: Human CLEC4E shares 65% and 68% amino acid sequence identity with mouse and rat CLEC4E in the extracellular domain, respectively . This moderate homology means that antibodies raised against one species may not recognize others equally well. Researchers must select antibodies validated for their species of interest .

• Functional divergence awareness: Research has revealed significant functional differences between human and mouse CLEC4E. For example, mouse CLEC4E does not appear to interact with TDB, GroMM, or cholesterol crystals, whereas human CLEC4E does . This functional divergence necessitates species-specific experimental designs and interpretation.

• Cross-reactivity validation protocols:

  • Test antibodies on recombinant proteins from multiple species

  • Validate on cell lines transfected with species-specific CLEC4E variants

  • Confirm specificity using tissues from knockout models of each species

• Epitope mapping across species:

  Antibody	Human Reactivity	Mouse Reactivity	Rat Reactivity	Optimal Applications
  sc-390806 (B-7)	Yes	Yes	Yes	WB, IP, IF, ELISA
  MAB8995	Yes	Limited	Limited	Flow cytometry
  Polyclonal antibodies	Variable	Variable	Variable	Dependent on immunogen

• Translation strategies for animal models:

  • When translating findings between species, validate key observations using multiple antibody clones

  • Consider using humanized mouse models for studies focusing on human-specific CLEC4E functions

  • Complement antibody studies with genetic approaches (e.g., CRISPR-mediated tagging) to overcome antibody limitations

Understanding and accounting for these species differences is crucial for generating reliable and translatable data in CLEC4E research across different model systems.

What methodological adaptations are needed when comparing CLEC4E expression between human and rodent samples?

Comparing CLEC4E expression between human and rodent samples requires specific methodological adaptations:

• Antibody selection strategy:

  • Use antibodies validated for cross-species reactivity, such as the B-7 clone (sc-390806) that detects CLEC4E from mouse, rat, and human origins

  • For species-specific detection, select antibodies targeting highly conserved epitopes or use species-specific antibodies in parallel experiments

  • Validate antibody performance on each species using positive and negative controls before comparative studies

• Normalization approaches:

  • Implement species-specific housekeeping proteins for western blot normalization

  • Use species-matched isotype controls for flow cytometry studies

  • For qPCR analysis, design primers for conserved regions or use species-specific primers with similar amplification efficiencies

• Sample preparation considerations:

  • Optimize tissue dissociation protocols separately for human and rodent tissues

  • Standardize cell isolation procedures to ensure comparable cell populations

  • Account for differences in autofluorescence between species in flow cytometry and immunofluorescence applications

• Data interpretation framework:

  • Acknowledge the 67% sequence identity between human and mouse CLEC4E when interpreting quantitative differences

  • Consider the biological context when comparing expression levels (e.g., specific pathogen responses may differ)

  • Focus on relative changes within each species rather than absolute expression values between species

• Functional validation:

  • Complement expression studies with functional assays tailored to species-specific CLEC4E ligands

  • Account for differences in ligand recognition (e.g., mouse CLEC4E doesn't recognize TDB, GroMM, or cholesterol crystals)

  • Use genetic approaches (knockdown/knockout) to confirm specificity of observed differences

These methodological adaptations enable meaningful cross-species comparisons while acknowledging the inherent differences in CLEC4E biology between humans and rodents.

How can researchers effectively employ CLEC4E antibodies in single-cell analysis technologies?

Employing CLEC4E antibodies in single-cell analysis requires specialized approaches for different platforms:

• Mass cytometry (CyTOF) applications:

  • Conjugate anti-CLEC4E antibodies with rare earth metals (e.g., lanthanides) for mass cytometry

  • Titrate metal-conjugated antibodies carefully to optimize signal-to-noise ratio

  • Include CLEC4E in panels with other C-type lectin receptors and myeloid markers for comprehensive phenotyping

  • Implement barcoding strategies to minimize batch effects when comparing multiple conditions

• Single-cell RNA-seq with protein detection (CITE-seq):

  • Modify anti-CLEC4E antibodies with oligonucleotide tags for CITE-seq applications

  • Balance antibody concentration to avoid oversaturation of the system

  • Design panels that capture CLEC4E alongside its known signaling partners for correlation analysis

  • Analyze data for correlation between CLEC4E protein levels and transcript expression

• Imaging mass cytometry for spatial context:

  • Validate metal-conjugated CLEC4E antibodies on control tissues before application

  • Combine with tissue microenvironment markers to assess spatial relationships

  • Implement computational approaches to quantify CLEC4E+ cell distributions relative to pathological features

  • Correlate with standard immunohistochemistry for validation

• Flow cytometry-based single-cell sorting:

  • Use bright fluorophore-conjugated anti-CLEC4E antibodies (e.g., PE or APC) for index sorting

  • Implement compensation controls specific for each fluorophore combination

  • Design gating strategies that account for CLEC4E expression heterogeneity

  • Consider density of CLEC4E expression when setting sorting parameters

• Analytical considerations:

  • Apply dimensionality reduction techniques (t-SNE, UMAP) to visualize CLEC4E+ populations

  • Implement clustering algorithms to identify novel CLEC4E+ subpopulations

  • Correlate CLEC4E expression with functional parameters at single-cell resolution

  • Use trajectory analysis to track CLEC4E expression changes during cell differentiation or activation

These approaches enable researchers to leverage single-cell technologies for nuanced investigation of CLEC4E biology across heterogeneous cell populations and tissue contexts.

What are the optimal approaches for studying CLEC4E-ligand interactions using antibody-based techniques?

Studying CLEC4E-ligand interactions requires specialized antibody-based techniques:

• Proximity ligation assays (PLA):

  • Combine anti-CLEC4E antibodies with antibodies against putative ligands (e.g., SAP130, TDM)

  • Implement in situ PLA on cells exposed to different stimulation conditions

  • Quantify interaction signals using confocal microscopy and specialized image analysis

  • Compare signal patterns between different cell activation states

• Förster resonance energy transfer (FRET):

  • Label anti-CLEC4E antibodies with donor fluorophores and potential ligands with acceptor fluorophores

  • Monitor energy transfer as a measure of molecular proximity (<10 nm)

  • Implement time-resolved FRET to capture dynamic interaction kinetics

  • Use flow cytometry-based FRET for population-level quantification

• Antibody-based blocking studies:

  • Apply anti-CLEC4E antibodies that target the carbohydrate recognition domain to block ligand binding

  • Compare multiple antibody clones for differential blocking effects on various ligands

  • Quantify downstream signaling events (e.g., phosphorylation of Syk, CARD9) as functional readouts

  • Implement dose-response studies to determine ligand-specific binding affinities

• Pull-down assays with antibody-conjugated beads:

  • Use agarose-conjugated CLEC4E antibodies (e.g., sc-390806 AC) for immunoprecipitation

  • Implement cross-linking strategies to capture transient interactions

  • Apply mass spectrometry to identify novel binding partners

  • Validate interactions using reciprocal immunoprecipitation approaches

• Surface plasmon resonance with antibody-based detection:

  • Immobilize CLEC4E ligands on sensor chips

  • Apply cells expressing CLEC4E followed by fluorophore-conjugated anti-CLEC4E antibodies

  • Quantify binding kinetics under different conditions (Ca²⁺ dependency, pH sensitivity)

  • Compare binding profiles between human and rodent CLEC4E to identify species-specific interactions

These sophisticated approaches enable detailed characterization of CLEC4E-ligand interactions, providing insights into receptor specificity, binding kinetics, and the structural basis of recognition events.

How should researchers interpret CLEC4E antibody signals in the context of heterogeneous myeloid populations?

Interpreting CLEC4E antibody signals in heterogeneous myeloid populations requires nuanced approaches:

• Multi-parameter analysis framework:

  • Always combine CLEC4E staining with lineage-defining markers (CD14, CD16, CD11c, etc.)

  • Implement dimensionality reduction techniques like t-SNE or UMAP to visualize relationships

  • Quantify CLEC4E expression as both percentage of positive cells and mean fluorescence intensity within defined subpopulations

  • Compare expression across activation states using standardized gating strategies

• Contextual variation considerations:

  • Recognize that CLEC4E expression is dynamically regulated and can vary substantially based on:

    • Differentiation state of myeloid cells

    • Inflammatory microenvironment

    • Tissue residence vs. circulation

    • Disease context (infection, autoimmunity, cancer)

• Technical interpretation guidelines:

  Myeloid Population	Expected CLEC4E Expression	Common Artifacts	Validation Approach
  Classical monocytes	Moderate to high	Fc receptor binding	Fc block essential
  Macrophages	Variable, activation-dependent	Autofluorescence	Unstained controls
  Dendritic cells	Lower, subset-dependent	Non-specific uptake	Isotype controls
  Neutrophils	Minimal/absent	High background	Multiple antibody validation

• Single-cell resolution analysis:

  • Implement imaging flow cytometry to correlate CLEC4E localization with expression level

  • Use single-cell RNA-seq with antibody tags (CITE-seq) to correlate protein expression with transcriptional state

  • Apply trajectory analysis to map CLEC4E expression changes during myeloid cell differentiation or activation

• Functional correlation approaches:

  • Correlate CLEC4E expression levels with functional responses to known ligands

  • Implement cell sorting based on CLEC4E expression followed by functional assays

  • Consider ratio of CLEC4E to other pattern recognition receptors rather than absolute expression alone

These interpretation frameworks enable researchers to extract meaningful biological insights from CLEC4E expression patterns across diverse and dynamic myeloid populations.

What analytical approaches help distinguish between specific and non-specific binding of CLEC4E antibodies?

Distinguishing specific from non-specific binding of CLEC4E antibodies requires rigorous analytical approaches:

• Comprehensive blocking strategy validation:

  • Implement titrated blocking protocols using:

    • Fc receptor blocking reagents (critical for myeloid cells)

    • Serum matching the species of secondary antibody

    • Non-fat dry milk or BSA for hydrophobic interactions

  • Compare signal reduction between target cells and known negative cells to optimize blocking conditions

• Peptide competition analysis:

  • Pre-incubate CLEC4E antibodies with excess immunizing peptide

  • Compare staining patterns with and without peptide competition

  • Quantify signal reduction as percentage of specific binding

  • Use non-relevant peptides as controls to confirm specificity of competition

• Signal distribution analysis:

  • Analyze subcellular localization patterns consistent with CLEC4E biology:

    • Membrane-predominant staining for surface CLEC4E

    • Golgi-associated staining for newly synthesized CLEC4E

    • Endosomal localization after ligand-induced internalization

  • Non-specific binding typically shows different distribution patterns (diffuse cytoplasmic, nuclear, etc.)

• Statistical approaches to signal discrimination:

  • Implement signal-to-noise ratio calculations across different antibody concentrations

  • Apply coefficient of variation analysis to assess staining consistency

  • Use receiver operating characteristic (ROC) curve analysis to determine optimal positive/negative thresholds

  • Employ machine learning algorithms to distinguish staining patterns

• Cross-validation with orthogonal techniques:

  • Correlate antibody-based detection with mRNA expression data

  • Compare multiple antibody clones recognizing different epitopes

  • Validate specificity using genetic approaches (siRNA knockdown, CRISPR knockout)

  • Implement western blot analysis to confirm molecular weight specificity

These analytical approaches provide a robust framework for distinguishing genuine CLEC4E signals from technical artifacts, ensuring reliable interpretation of experimental results across different applications.