Fpr2 Antibody

What is FPR2 and why is it significant in research?

FPR2 (Formyl Peptide Receptor 2) is a G-protein coupled receptor with seven transmembrane domains, predominantly located on the surface of phagocytic leukocytes such as neutrophils and monocytes. It's also known by several other names including ALXR, FPRL1, FMLP-R-II, FMLPX, FPR2A, and N-formyl peptide receptor 2 .

FPR2 is significant in research because it:

• Plays a crucial role in mediating inflammatory responses and host defense mechanisms

• Binds to various ligands, including lipoxin A4 and N-formyl-methionyl-leucyl-phenylalanine (fMLP)

• Triggers cellular migration and calcium mobilization upon activation

• Has been implicated in various pathological conditions, including Alzheimer's disease and depression

• Has both pro-inflammatory and pro-resolving roles, making it an intriguing target for immunomodulatory therapies

The protein is approximately 39 kilodaltons in mass and is expressed in various cell types, including peripheral immune cells as well as central microglia and neurons .

What are the primary applications of FPR2 antibodies in neuroscience research?

FPR2 antibodies have emerged as valuable tools in neuroscience research, particularly for studying neuroinflammation and neurodegenerative conditions:

Key Applications in Neuroscience:

• Detecting FPR2 expression in microglia: FPR2 antibodies are crucial for identifying and quantifying FPR2-expressing microglia, particularly capillary-associated microglia (CAMs) in the brain

• Studying blood-brain barrier (BBB) integrity: FPR2 plays a role in BBB function, and antibodies help assess how FPR2 modulation affects barrier integrity

• Investigating neuroinflammatory processes: FPR2 antibodies are used to examine the role of this receptor in microglial activation and neuroinflammation

• Alzheimer's disease research: FPR2 interacts with amyloid beta peptides and has been implicated in phagocyte attraction to sites of amyloid plaques

• Depression models: Recent research has demonstrated FPR2 upregulation in social isolation-induced depression models, making FPR2 antibodies important for studying mood disorders

In a recent study, immunofluorescence staining with anti-FPR2 antibodies revealed that social isolation leads to an increase in FPR2-expressing CAMs in the prefrontal cortex and hippocampus of mice, indicating FPR2's potential involvement in depression pathophysiology .

How do I select the most appropriate FPR2 antibody for my flow cytometry experiments?

Selecting the optimal FPR2 antibody for flow cytometry requires careful consideration of several factors:

Selection Criteria:

Practical Application Guidance:

• For human peripheral blood monocytes, clone 304405 (available as fluorescein or APC-conjugated) has been validated and shown effective in distinguishing positive and negative populations

• When staining membrane-associated FPR2, follow established protocols for membrane protein detection, such as those provided by R&D Systems

• Consider co-staining with other markers (e.g., CD14 for monocytes) to properly identify FPR2-expressing cell populations

What methodological approaches are recommended for detecting FPR2 in brain tissue samples?

Detection of FPR2 in brain tissue requires specialized immunofluorescence techniques:

Recommended Protocol for Brain Tissue Immunofluorescence:

• Tissue preparation:

  • Perfuse animals with PBS followed by 4% paraformaldehyde

  • Post-fix brain tissue and prepare sections at appropriate thickness (20-40 μm)

• Immunofluorescence staining sequence:

  • Rinse sections with 0.05 M TBS

  • Permeabilize with 0.1% Triton X-100 in TBS for 10 minutes

  • Block with 5% normal donkey serum in TBS (0.1% Tween-20) for 1 hour at room temperature

• Triple staining approach for FPR2 in CAMs:

  • First incubate with anti-CD31 (brain endothelial cell marker) antibody (1:200) overnight at 4°C

  • Wash and apply AlexaFluor-555-conjugated anti-rat antibody (1:500) for 1 hour

  • Incubate with anti-Iba1 (microglial marker) antibody (1:200) overnight

  • Wash and apply AlexaFluor-488-conjugated anti-rabbit antibody (1:500) for 1 hour

  • Incubate with anti-FPRL1/AF647 antibody (1:200) for 1 hour at room temperature

  • Counterstain with DAPI for 10 minutes before mounting

• Quantification methods:

  • For colocalization analysis, use Manders' colocalization coefficients calculated via the Coloc2 plugin of Fiji software

  • The M2 coefficient represents the ratio of fluorescence intensity of Iba1 overlapping with FPR2 to the total FPR2 intensity

This approach allows for precise detection of FPR2-expressing microglia associated with blood vessels, which is critical for studying neuroinflammatory processes in various neurological conditions.

What are the key differences between monoclonal and polyclonal FPR2 antibodies for research applications?

Understanding the differences between monoclonal and polyclonal FPR2 antibodies is crucial for selecting the appropriate tool:

Comparative Analysis:

Selection guidance:

• For precise mapping of specific FPR2 epitopes, monoclonal antibodies are preferred

• For detection of FPR2 in fixed tissues or under denaturing conditions, polyclonal antibodies may provide better sensitivity

• When reproducibility between experiments is critical, monoclonal antibodies offer more consistent results

• For novel applications or unstudied species, polyclonal antibodies might provide higher chances of recognition

How can I troubleshoot weak or inconsistent FPR2 antibody staining in flow cytometry?

Flow cytometry with FPR2 antibodies can present several technical challenges. Here's a systematic troubleshooting approach:

Common Issues and Solutions:

• Weak or absent signal:

  • Cause: Insufficient FPR2 expression or antibody concentration

  • Solution: Verify FPR2 expression in your cell type; use positive controls like human peripheral blood monocytes

  • Method: Increase antibody concentration in a titration experiment (typically 1:50 to 1:200 dilution range)

• High background:

  • Cause: Non-specific binding or autofluorescence

  • Solution: Optimize blocking (5-10% serum from the same species as secondary antibody)

  • Method: Include proper isotype controls (e.g., Catalog # IC0041A for APC conjugates)

• Inconsistent results between experiments:

  • Cause: Variability in cell preparation or staining conditions

  • Solution: Standardize protocols for cell isolation and staining

  • Method: Use calibration beads to normalize instrument settings between experiments

• Poor discrimination between positive and negative populations:

  • Cause: Suboptimal antibody clone or fluorophore choice

  • Solution: Test validated clones like 304405 with bright fluorophores (APC preferable to FITC for dim antigens)

  • Method: Examine published flow cytometry histograms showing clear population separation

• Cell viability issues affecting staining:

  • Cause: Dead/dying cells bind antibodies non-specifically

  • Solution: Include viability dye and gate on live cells only

  • Method: Follow protocols specifically for staining membrane-associated proteins

For optimal results, consider using standardized protocols such as R&D Systems' method for staining membrane-associated proteins, which has been validated for FPR2 detection in human monocytes .

What is the significance of FPR2 in neuroinflammation and how can antibodies help investigate this?

FPR2 plays a complex role in neuroinflammation, and antibodies are essential tools for investigating these mechanisms:

FPR2's Role in Neuroinflammation:

• Functions as a dual-activity receptor that can mediate both pro-inflammatory and pro-resolving responses

• Expressed predominantly in microglia, particularly capillary-associated microglia (CAMs) in the brain

• Interacts with formyl peptides to attract phagocytes to sites of infection and promote inflammatory reactions

• Also binds to amyloid beta peptides, potentially contributing to microglial responses in Alzheimer's disease

• Expression increases in response to neuroinflammatory stimuli and social isolation stress

Research Applications of FPR2 Antibodies:

• Mapping expression patterns:

  • FPR2 antibodies enable visualization of receptor distribution in different brain regions

  • Research has shown FPR2 upregulation in the prefrontal cortex and hippocampus during stress conditions

• Investigating microglial-vascular interactions:

  • Using FPR2 antibodies in combination with microglial (Iba1) and endothelial (CD31) markers reveals that:

    • Social isolation increases CAMs with upregulated FPR2 expression

    • FPR2 colocalized with Iba1 accounts for approximately 66-77% of total FPR2 expression in normal conditions

    • This proportion increases to 74-83% during social isolation stress

• Evaluating therapeutic interventions:

  • FPR2 antibodies can assess how antagonists (like WRW4) affect receptor expression and microglial activation

  • Studies have shown that FPR2 antagonism reduces microglial activation and neuronal damage in stress models

• Blood-brain barrier research:

  • FPR2 antibodies help investigate the relationship between receptor activation and BBB integrity

  • Recent findings suggest FPR2 antagonism can restore compromised BBB function

These applications demonstrate how FPR2 antibodies serve as critical tools for understanding neuroinflammatory processes and developing potential therapeutic strategies for neurological and psychiatric disorders.

How do different fixation and permeabilization protocols affect FPR2 antibody performance in immunofluorescence applications?

Fixation and permeabilization methods significantly impact FPR2 antibody performance in immunofluorescence staining:

Fixation Method Comparison:

Permeabilization Optimization:

For FPR2 detection in brain tissue, the following permeabilization protocol has been validated:

• 0.1% Triton X-100 in TBS for 10 minutes at room temperature

• Alternative: 0.1-0.3% Tween-20 for more gentle permeabilization

Critical Considerations:

• Epitope accessibility: FPR2 is a seven-transmembrane protein, so epitope accessibility varies with antibody clone and fixation

• Antibody validation: Always verify that your chosen antibody has been validated with your specific fixation method

• Signal amplification: For low-abundance FPR2 detection, consider tyramide signal amplification systems

• Blocking optimization: Using 5% normal donkey serum in TBS (0.1% Tween-20) for 1 hour at room temperature effectively reduces background

For optimal results with FPR2 antibodies in neuronal tissues, the protocol used in recent studies involving triple-staining of FPR2, CD31, and Iba1 provides a well-validated approach with demonstrated success in visualizing FPR2-expressing microglia associated with blood vessels .

What are the most advanced techniques for quantifying FPR2 expression in immune and neural cells?

Advanced quantification of FPR2 requires sophisticated approaches beyond simple detection:

State-of-the-Art Quantification Techniques:

• Flow Cytometry with Spectral Analysis:

  • Enables precise quantification of FPR2 receptor density using antibody binding capacity (ABC) beads

  • Allows multiple parameter analysis (10+ colors) to correlate FPR2 expression with other markers

  • Facilitates identification of FPR2+ cell subpopulations within heterogeneous samples

  • Validated FPR2 antibodies like clone 304405 provide reliable detection in human monocytes

• Confocal Microscopy with Colocalization Analysis:

  • Manders' colocalization coefficients quantify the degree of overlap between FPR2 and cellular markers

  • M2 coefficient: ratio of Iba1 fluorescence overlapping with FPR2 to total FPR2 fluorescence

  • This approach revealed that FPR2 colocalized with Iba1 accounts for 66-77% of total FPR2 expression in normal conditions and increases to 74-83% during stress

• ImageJ-Based Quantification Methods:

  • For tissue sections, relative immunofluorescence intensities of FPR2 can be precisely measured

  • Microglial activation can be assessed by delineating cell bodies and quantifying area changes

  • The Coloc2 plugin provides standardized colocalization analysis

• Single-Cell RNAseq Correlated with Protein Expression:

  • Correlating FPR2 transcript levels with protein expression measured by antibodies

  • Enables identification of cell-specific regulation patterns

  • Recent transcriptomic analyses have revealed FPR2 upregulation in specific brain regions in depression models

• Super-Resolution Microscopy:

  • Techniques like STORM or PALM with FPR2 antibodies enable nanoscale receptor localization

  • Reveals receptor clustering and membrane distribution patterns not visible with conventional microscopy

These advanced techniques provide deeper insights into FPR2 biology by not only detecting presence/absence but quantifying expression levels, cellular distribution, and colocalization with other proteins across different experimental conditions.

How does FPR2 expression change in neuropsychiatric disorders and what are the implications for antibody-based detection methods?

Recent research has uncovered significant alterations in FPR2 expression in neuropsychiatric conditions:

FPR2 Expression Changes in Neuropsychiatric Disorders:

Implications for Antibody-Based Detection:

• Sensitivity requirements:

  • Subtle changes in FPR2 expression levels require highly sensitive antibodies

  • Quantitative rather than merely qualitative detection becomes essential

• Cell type-specific detection challenges:

  • FPR2 upregulation may be cell-type specific (e.g., primarily in microglia)

  • Multi-label approaches combining FPR2 antibodies with cell-type markers are necessary

  • Example: Triple staining with anti-FPR2, anti-Iba1 (microglia), and anti-CD31 (endothelial cells)

• Method modifications for pathological samples:

  • Increased background in inflamed tissues may require modified blocking protocols

  • Autofluorescence quenching may be necessary for certain tissue types

  • Careful selection of antibody clones validated in pathological samples is critical

• Translational considerations:

  • When comparing animal models to human samples, species cross-reactivity of antibodies must be considered

  • FPR2 antibodies with validated reactivity to both human and rodent proteins are valuable for translational research