ADGRE1 Antibody

What is ADGRE1 and what cell types express it across different species?

ADGRE1 (Adhesion G Protein-Coupled Receptor E1), also known as EMR1 or F4/80, is a surface receptor belonging to the adhesion family of G protein-coupled receptors. Its structure includes seven transmembrane domains, an intracellular C-terminus, and an extracellular N-terminus containing six EGF-like domains . The expression pattern of ADGRE1 varies significantly between species:

In humans, ADGRE1 is exclusively expressed on mature eosinophils in the blood, bone marrow, and nasal polyps, making it a potential therapeutic target for eosinophilic disorders . In mice, the F4/80 protein (encoded by the Adgre1 locus) is widely used as a marker for murine macrophage populations . In pigs, ADGRE1 functions as a myeloid differentiation marker, absent from progenitors in bone marrow but highly expressed in mature granulocytes, monocytes, and tissue macrophages .

RNA-Seq analysis has confirmed ADGRE1 mRNA expression in macrophages across multiple mammalian species including pig, human, rat, sheep, goat, cow, water buffalo, and horse, though with inter-species variation in expression levels and response to stimulation .

How do I select the appropriate ADGRE1 antibody for my experimental system?

Selecting the right ADGRE1 antibody requires careful consideration of several factors:

• Species reactivity: Ensure the antibody recognizes ADGRE1 in your target species. For example, anti-F4/80 antibodies like Boster Bio's A08751 are specific to mouse and rat ADGRE1 , while Alomone Labs' AER-051-F antibody recognizes human EMR1 .

• Application compatibility: Verify the antibody has been validated for your specific application (IHC, flow cytometry, Western blot, etc.). Some antibodies, like the anti-F4/80 antibody from Boster, are tested specifically for immunofluorescence and immunohistochemistry applications .

• Epitope location: For intact cell applications, choose antibodies targeting extracellular domains. The Anti-EMR1 (ADGRE1) extracellular-FITC antibody from Alomone Labs specifically targets the extracellular N-terminus (amino acid residues 58-72 of human EMR1), making it ideal for detecting the receptor on living cells .

• Clonality: Polyclonal antibodies often provide stronger signals but may have higher background, while monoclonal antibodies offer greater specificity. The mouse anti-pig ADGRE1 (ROS-4E12-3E6) is a monoclonal antibody developed specifically for high specificity .

• Validation data: Review available validation data in tissues or cells relevant to your research. The anti-F4/80 antibody (A08751) has been validated on mouse and rat samples with no cross-reactivity reported with other proteins .

What are the optimal sample preparation protocols for ADGRE1 immunostaining?

Optimal sample preparation for ADGRE1 immunostaining varies based on the application and tissue type:

For frozen sections:

• Place tissues in phosphate-buffered saline (pH 7.0) and mount in OCT compound

• Cut 10 µm sections and mount on Superfrost slides

• Dry for 24 hours at 4°C before use

• Fix in ice-cold methanol for 10 minutes

• Wash twice (5 minutes each) in PBS

• Block endogenous peroxidase activity with 0.3% hydrogen peroxide in PBS

• Wash twice in PBS (5 minutes each)

• Block with Tris-buffered saline containing 20% normal goat serum and 5% BSA for 1 hour at room temperature

For live cell immunostaining:

• Collect live intact cells (e.g., mouse J774 macrophage cells or human THP-1 monocytic leukemia cells)

• Include appropriate controls (untreated cells and isotype control-FITC)

• Add Anti-EMR1 antibody (e.g., 2.5 μg of Anti-EMR1-FITC from Alomone Labs) to visualize cell surface expression

For FFPE tissues, standard antigen retrieval protocols should be employed, though specific conditions may need optimization based on fixation time and antibody specifications.

How can I use ADGRE1 antibodies to differentiate macrophage subpopulations?

ADGRE1 antibodies can be powerful tools for differentiating macrophage subpopulations when used in combination with other markers and appropriate analytical techniques:

• Multi-parameter flow cytometry: Combine ADGRE1 antibodies with other macrophage markers such as CD163. In pigs, for example, dual staining with anti-ADGRE1 (ROS-4E12-3E6) and anti-CD163 antibodies has been used for characterizing tissue macrophage populations . Design a panel that includes:

  • ADGRE1 for general macrophage identification

  • CD163 for M2-like/anti-inflammatory macrophages

  • CD80/CD86 for M1-like/pro-inflammatory macrophages

  • Tissue-specific markers as needed

• Sequential immunohistochemistry: For tissue sections where co-localization analysis is desired:

  • Perform ADGRE1 staining with 3,3'-diaminobenzidine (DAB) detection

  • Follow with a second macrophage marker using a different chromogen

  • This allows visualization of macrophage heterogeneity within tissue microenvironments

• Quantitative analysis: Implement digital image analysis to quantify:

  • The ratio of ADGRE1+ to ADGRE1- macrophages

  • Co-expression levels of ADGRE1 with other markers

  • Spatial distribution patterns of different macrophage subsets

• In vivo stimulation studies: ADGRE1 expression can be enhanced by CSF1 (macrophage colony-stimulating factor) treatment, which increases both the number of macrophages and the level of ADGRE1 expression on individual cells . This property can be utilized to study macrophage dynamics in response to stimuli.

What controls should I include when validating a new ADGRE1 antibody lot?

Proper validation of ADGRE1 antibodies requires comprehensive controls to ensure specificity and reproducibility:

• Positive tissue controls:

  • For human studies: Tissues rich in eosinophils (e.g., nasal polyps)

  • For mouse/rat studies: Macrophage-rich tissues (e.g., spleen, liver, especially after CSF1 treatment)

  • Cell lines with known ADGRE1 expression (e.g., J774 macrophages for mouse, THP-1 for human)

• Negative controls:

  • Isotype control antibody (e.g., rabbit IgG isotype control-FITC for Anti-EMR1-FITC antibodies)

  • Tissues known to lack ADGRE1 expression

  • Primary antibody omission controls

  • Cell lines that do not express ADGRE1

• Blocking experiments:

  • Pre-incubation of antibody with immunizing peptide (e.g., the peptide CKQGFLSSNGQNHFK for Alomone Labs' Anti-EMR1 antibody)

  • Concentration-dependent reduction in signal confirms specificity

• Cross-reactivity assessment:

  • Test antibody on tissues from multiple species if cross-reactivity is claimed

  • Verify absence of binding to related proteins (particularly important for other ADGRE family members)

• Lot-to-lot comparison:

  • Direct comparison with previously validated lot on identical samples

  • Statistical analysis of staining intensity and pattern

• Orthogonal validation:

  • Correlation with mRNA expression data

  • Comparison with alternative antibody clones targeting different epitopes

How do fixation methods affect ADGRE1 detection in immunohistochemistry?

Fixation methods can significantly impact ADGRE1 detection due to potential epitope masking or destruction:

• Methanol fixation: Often preferred for ADGRE1 immunostaining as demonstrated in protocols using mouse anti-pig ADGRE1 antibodies. Ice-cold methanol for 10 minutes preserves antigenicity while providing adequate fixation for frozen sections .

• Paraformaldehyde fixation: May require optimization of antigen retrieval methods:

  • Heat-induced epitope retrieval in citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • Enzymatic retrieval using proteinase K may be effective for some antibody clones

  • Duration of fixation should be minimized (4-24 hours optimal)

• Unfixed/live cell applications: For cell surface epitopes, live cell staining may provide superior results, particularly when using antibodies targeting extracellular domains such as Alomone Labs' Anti-EMR1 (ADGRE1) (extracellular)-FITC Antibody .

• Acetone fixation: May offer a good compromise for frozen sections when methanol fixation is suboptimal.

• Comparison of fixation methods for ADGRE1 detection:

What are common causes of false negative results with ADGRE1 antibodies?

Several factors can contribute to false negative results when using ADGRE1 antibodies:

• Epitope masking: The large EGF-like domains in ADGRE1's extracellular region may be particularly susceptible to conformational changes during fixation. Consider:

  • Testing multiple fixation protocols (e.g., comparing methanol, paraformaldehyde, and acetone)

  • Optimizing antigen retrieval methods (heat-induced vs. enzymatic)

  • Using antibodies targeting different epitopes (N-terminal vs. C-terminal)

• Species-specific variations: ADGRE1 shows substantial sequence variation between species with evidence of rapid evolution . Ensure:

  • The antibody has been validated for your specific species

  • If cross-species reactivity is claimed, verify with appropriate controls

  • Consider species-specific structural variations like the duplication of EGF-like domains seen in ruminants and horses

• Alternative splicing and exon skipping: RNA-Seq data reveals evidence of exon skipping of the EGF-like domains in ADGRE1 across multiple species . This may result in:

  • Epitope absence in certain cell populations

  • Variable antibody binding even within the same tissue

  • Consider using antibodies targeting constant regions of the protein

• Technical issues:

  • Insufficient antibody concentration

  • Inadequate incubation time (consider overnight incubation at 4°C for improved signal)

  • Degraded antibody (verify storage conditions and expiration dates)

  • Excessive washing (optimize washing steps without compromising specificity)

• Low expression levels: In some tissues or conditions, ADGRE1 expression may be below detection thresholds. Consider:

  • Signal amplification methods (e.g., tyramide signal amplification)

  • More sensitive detection systems (e.g., switching from chromogenic to fluorescent detection)

  • Pre-enrichment of target cells before analysis

How can I optimize dual immunostaining protocols involving ADGRE1?

Optimizing dual immunostaining with ADGRE1 antibodies requires careful attention to antibody compatibility and protocol design:

• Primary antibody selection:

  • Choose antibodies raised in different host species (e.g., mouse anti-ADGRE1 and rabbit anti-CD163)

  • If same-species antibodies must be used, consider directly conjugated antibodies or sequential immunostaining

  • Verify that epitopes are sufficiently separated spatially to prevent steric hindrance

• Sequential vs. simultaneous staining:

  • Sequential: Complete the entire staining protocol for one antibody before beginning the second

  • Simultaneous: Incubate both primary antibodies together, then both secondary antibodies

  • For ADGRE1, sequential staining often provides cleaner results, especially when one marker is more abundant than the other

• Signal separation strategies:

  • Chromogenic: Use contrasting chromogens (e.g., DAB for ADGRE1 and Fast Red for second marker)

  • Fluorescent: Select fluorophores with minimal spectral overlap

  • Include appropriate single-stained controls to verify absence of cross-reactivity

• Blocking optimization:

  • When using mouse anti-pig ADGRE1 on pig tissues, include additional blocking steps to reduce endogenous Fc receptor binding

  • Consider using fragment antibodies (Fab) rather than whole IgG to reduce non-specific binding

  • Add species-specific serum matching the host species of secondary antibodies

• Validated dual staining combinations:

  • ADGRE1 + CD163: Demonstrated in pig tissues using ROS-4E12-3E6 and anti-human CD163

  • For mouse tissues: F4/80 + CD11b or F4/80 + CD68 provide complementary macrophage identification

• Troubleshooting dual staining issues:

  • If signal crosstalk occurs, increase washing steps between protocol stages

  • Consider tyramide signal amplification for the weaker marker

  • Test antibody concentrations individually before combining to establish optimal working dilutions

How do post-translational modifications affect ADGRE1 antibody binding?

Post-translational modifications (PTMs) of ADGRE1 can significantly impact antibody binding and may explain variability in staining patterns:

• Glycosylation:

  • ADGRE1 contains a serine/threonine-rich domain reminiscent of mucin-like glycoproteins

  • Heavy glycosylation may mask epitopes, particularly in the extracellular domain

  • Consider testing enzymatic deglycosylation before staining to improve antibody access

  • Select antibodies targeting regions less likely to be affected by glycosylation

• Proteolytic cleavage:

  • As an adhesion GPCR, ADGRE1 may undergo autoproteolysis at the GPS (GPCR proteolysis site)

  • This creates separate N-terminal and C-terminal fragments that remain non-covalently associated

  • Antibodies targeting epitopes near the cleavage site may show variable binding

  • Consider using antibodies that target regions distant from known cleavage sites

• Phosphorylation:

  • Intracellular domains may be phosphorylated during receptor activation

  • This can affect binding of antibodies targeting cytoplasmic domains

  • For functional studies, phospho-specific antibodies may be valuable

  • For general detection, extracellular epitopes are less affected by phosphorylation status

• Conformational changes:

  • Ligand binding may induce conformational changes in ADGRE1

  • This may expose or mask certain epitopes

  • Consider using multiple antibodies targeting different regions for comprehensive detection

  • Fixation methods may differentially preserve these conformational states

• Strategies to address PTM interference:

  • Use a panel of antibodies targeting different domains

  • Compare native versus denatured detection methods

  • Consider native immunoprecipitation followed by Western blotting under reducing conditions

  • Always include positive controls with known PTM status

How can ADGRE1 antibodies be used to study macrophage recruitment in disease models?

ADGRE1 antibodies offer valuable tools for investigating macrophage dynamics in various disease models:

• Temporal analysis of macrophage infiltration:

  • Use ADGRE1 immunostaining at different time points to track macrophage recruitment

  • CSF1 treatment has been shown to induce ADGRE1 expression in liver macrophages in multiple species, providing a model system for studying macrophage recruitment

  • Quantify both cell numbers and intensity of ADGRE1 expression per cell to track maturation state

• Tissue-specific macrophage responses:

  • Compare ADGRE1+ cell infiltration across different tissues in systemic disease models

  • Combine with tissue-specific macrophage markers to distinguish resident versus recruited populations

  • Use digital pathology tools to analyze spatial distribution of macrophages relative to pathological features

• Therapeutic intervention assessment:

  • Evaluate changes in ADGRE1+ cell numbers and phenotype following experimental therapies

  • Given that human EMR1 is primarily expressed on eosinophils, it represents a potential therapeutic target for eosinophilic disorders

  • In mouse models, tracking F4/80+ macrophages can assess anti-inflammatory drug efficacy

• Flow cytometry applications:

  • Use Anti-EMR1 (extracellular)-FITC antibodies for quantifying circulating monocytes/eosinophils

  • Track changes in ADGRE1 expression intensity as a marker of activation/maturation

  • Combine with intracellular cytokine staining to correlate ADGRE1 expression with functional status

• Single-cell analysis approaches:

  • Sort ADGRE1+ cells for downstream transcriptomic or proteomic analysis

  • Correlate ADGRE1 expression levels with functional classification of macrophage subsets

  • Use ADGRE1 as a selection marker for macrophage-targeted single-cell RNA sequencing

What insights can ADGRE1 antibodies provide about species differences in macrophage biology?

ADGRE1 antibodies are valuable tools for comparative immunology research, highlighting important species differences:

• Expression pattern variations:

  • Human: ADGRE1 is predominantly expressed on mature eosinophils

  • Mouse: F4/80 (ADGRE1) is a widely used macrophage marker

  • Pig: ADGRE1 is expressed on macrophages, monocytes, and granulocytes

  • Using species-specific ADGRE1 antibodies allows comparative analysis of myeloid cell distribution

• Evolutionary insights:

  • RNA-Seq analysis reveals rapid evolution of ADGRE1 with significant inter-species variation

  • Ruminants and horses show complete duplication of the seven EGF-like domains

  • Evidence of exon skipping of EGF-like domains exists across species

  • Species-specific antibodies can help determine the functional significance of these evolutionary changes

• Response to stimulation:

  • Inter-species variation exists in ADGRE1 response to lipopolysaccharide (LPS) stimulation

  • CSF1 treatment induces ADGRE1 expression in rats, mice, and pigs

  • Species-comparative studies using ADGRE1 antibodies can reveal differential macrophage activation pathways

• Structural variations affecting antibody selection:

  • When designing cross-species studies, researchers must account for structural differences

  • Antibodies targeting highly conserved regions may provide cross-species reactivity

  • Validation is essential as even conserved epitopes may show differential accessibility

  • Multi-species analysis should include controls for each species tested

• Translational implications:

  • Understanding species-specific ADGRE1 biology is crucial for translating animal model findings to human applications

  • The distinct expression pattern in humans (eosinophils) versus mice (macrophages) has significant implications for interpreting mouse models of inflammatory diseases

  • ADGRE1 antibodies can help identify which species most closely resembles human macrophage biology for specific disease models

What are emerging applications of ADGRE1 antibodies in functional studies?

ADGRE1 antibodies are increasingly being used beyond simple detection for functional studies of macrophage/eosinophil biology:

• Receptor-mediated signaling studies:

  • Using antibodies to trigger ADGRE1 receptor activation

  • Monitoring downstream signaling events following antibody-mediated crosslinking

  • Comparing signaling outcomes between species to understand functional evolution of this receptor

• Cell depletion strategies:

  • Development of antibody-drug conjugates targeting ADGRE1

  • In human systems, ADGRE1-targeted depletion could provide eosinophil-specific therapy

  • In mouse models, F4/80-targeted approaches could achieve macrophage depletion

• Live cell imaging applications:

  • Anti-EMR1 (extracellular)-FITC antibodies are ideal for detecting the receptor on living cells

  • Time-lapse imaging of antibody-labeled cells to track macrophage/eosinophil dynamics

  • FRET-based approaches to study receptor interactions and conformational changes

• Targeted drug delivery:

  • ADGRE1 antibodies conjugated to nanoparticles for cell-specific delivery

  • Potential therapeutic targeting of eosinophils in human allergic disorders

  • Macrophage-targeted approaches in mouse models of inflammatory disease

• Functional blocking studies:

  • Development of antibodies that block ligand binding to ADGRE1

  • Assessment of functional outcomes following receptor blockade

  • Comparative studies across species to identify conserved functional domains

• Reporter systems:

  • Generation of recombinant ADGRE1 fusion proteins for functional studies

  • Creating antibody-based biosensors for ADGRE1 activation

  • Combination with CRISPR-engineered cell lines for mechanistic studies