P2A12 Antibody

What is the P2Y12 receptor and what cellular functions does it mediate?

The P2Y12 receptor belongs to the G-protein coupled receptor superfamily and functions primarily as a receptor for adenosine diphosphate (ADP) and adenosine triphosphate (ATP). It couples to G-proteins that inhibit the adenylyl cyclase second messenger system but is not activated by uridine diphosphate (UDP) or uridine triphosphate (UTP) .

The receptor plays essential roles in:

• Platelet activation and aggregation when co-expressed with P2Y1 receptor

• Normal blood coagulation processes

• Microglial cell function in the central nervous system

• Signal transduction pathways involving intracellular calcium mobilization

P2Y12 is a well-established therapeutic target for thromboembolism treatment and other clotting disorders, as its inhibition prevents excessive platelet aggregation .

Which tissue and cell types commonly express P2Y12 receptors?

P2Y12 receptor expression has been confirmed in multiple tissues and cell types using various detection methods:

• Platelets: Most abundant expression, critical for aggregation responses

• Brain: Predominantly in microglial cells, making it a useful microglial marker

• Peripheral tissues: Detected in lung, appendix, pituitary and adrenal glands

• Cell lines: Found in BV-2 microglia cells and rat basophilic leukemia (RBL) cells

When designing experiments targeting P2Y12, researchers should consider these expression patterns to select appropriate positive and negative control tissues .

What antibody formats are available for P2Y12 detection?

Several antibody formats have been developed for detecting P2Y12 in various experimental applications:

When selecting an antibody, researchers should consider the specific epitope recognition (e.g., extracellular domains), species cross-reactivity, and validation in the intended application .

What are recommended protocols for cell surface detection of P2Y12?

For successful cell surface detection of P2Y12 receptors on intact, living cells:

• Maintain cells in appropriate growth medium at proper density (typically 70-80% confluence)

• Harvest cells using a gentle detachment method that preserves surface epitopes (avoid harsh trypsinization)

• Resuspend cells in cold staining buffer (PBS with 1-2% serum or BSA)

• Incubate with primary anti-P2Y12 antibody at manufacturer's recommended dilution (typically 1:100-1:200) for 30-45 minutes at 4°C

• Wash cells 2-3 times with cold staining buffer

• Apply appropriate labeled secondary antibody (e.g., goat anti-rabbit-AlexaFluor-594)

• Incubate for 30 minutes at 4°C in darkness

• Wash thoroughly and analyze promptly

For flow cytometry applications specifically, use 5 μL of conjugated antibody per million cells in 100 μL staining volume or 5 μL per 100 μL of whole blood .

What controls should be included in P2Y12 antibody experiments?

Proper experimental controls are essential for rigorous P2Y12 antibody research:

• Positive tissue controls: Use rat brain membrane or human cerebrum tissue sections with known P2Y12 expression

• Negative controls: Include secondary antibody-only controls to assess background

• Blocking peptide controls: Pre-incubate antibody with P2Y12 receptor blocking peptide to confirm specificity

• Isotype controls: Use matched isotype antibodies (e.g., Mouse IgG2a, κ for some clones) to identify non-specific binding

• Knockout/knockdown controls: When available, use P2Y12-deficient samples to validate signal specificity

These controls help distinguish specific P2Y12 signal from background or non-specific binding, particularly important when examining tissues with low receptor expression levels .

How can P2Y12 antibodies be optimized for studying microglial activation in neuroinflammatory conditions?

Optimizing P2Y12 antibody protocols for microglia research requires careful consideration of several factors:

• Tissue preparation: For brain tissue immunohistochemistry, heat-mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes typically provides optimal epitope exposure

• Antibody selection: Choose antibodies validated specifically in microglial cells (e.g., BV-2 cell line)

• Dilution optimization: For paraffin-embedded human cerebrum tissue, dilutions of 1:40000 have been validated for some clones

• Co-staining approach:

  • Combine P2Y12 detection with additional microglial markers (Iba1, CD68)

  • Use multiplexed immunohistochemistry to simultaneously assess activation state markers

• Analysis methods: Employ quantitative image analysis to measure both receptor expression levels and morphological changes in microglia

P2Y12 expression typically decreases during microglial activation, making it a valuable marker for monitoring inflammatory responses in the CNS .

What methodological considerations are important when using P2Y12 antibodies to study platelet function?

When investigating platelet function with P2Y12 antibodies, researchers should implement these methodological considerations:

• Sample preparation:

  • Collect blood in anticoagulants that minimally affect platelet function (sodium citrate preferred)

  • Prepare platelet-rich plasma (PRP) promptly after collection

  • Consider testing both resting and activated platelets (using ADP stimulation)

• Functional assays:

  • Combine antibody detection with aggregometry to correlate receptor expression with function

  • When using P2Y12 antagonists like R-138727, assess dose-dependent inhibition of ADP-induced aggregation

  • Consider dual-pathway inhibition studies combining P2Y12 and GPIIb-IIIa antagonists

• Flow cytometric assessment:

  • Use forward/side scatter gating to identify platelet population

  • Consider dual staining with platelet marker CD41/CD61 for definitive identification

  • Analyze both receptor expression level (MFI) and percentage of positive platelets

• Data interpretation:

  • Account for inter-individual variability in receptor expression

  • Correlate with platelet activation markers (P-selectin, activated GPIIb-IIIa)

  • Analyze procoagulant activity parameters alongside receptor expression

These approaches enable comprehensive analysis of P2Y12 receptor in platelet function studies, particularly important when evaluating antiplatelet therapies .

How do P2Y12 receptor antagonists affect antibody binding in experimental settings?

The interaction between P2Y12 antagonists and antibody binding presents important experimental considerations:

• Epitope accessibility:

  • Small molecule antagonists (thienopyridines, direct P2Y12 inhibitors) may alter receptor conformation

  • Extracellular epitope-targeting antibodies may show reduced binding when antagonists occupy binding sites

  • Intracellular domain antibodies generally remain unaffected by antagonist binding

• Receptor internalization effects:

  • Some antagonists promote receptor internalization, reducing surface detection

  • Time-course experiments can distinguish between occupancy and downregulation

  • Consider fixed versus live cell detection protocols based on research question

• Experimental approach recommendations:

  • Include antagonist-free controls when studying antibody binding

  • Consider measuring total receptor (permeabilized cells) versus surface receptor

  • Use concentration-response curves to detect subtle antagonist effects on antibody binding

  • For in vivo studies, collect samples at multiple timepoints relative to antagonist administration

Understanding these interactions is crucial for accurate interpretation of experimental results, particularly in pharmacological studies evaluating P2Y12 inhibition in platelet function .

What approaches can improve specificity when using P2Y12 antibodies in complex tissue samples?

Enhancing P2Y12 antibody specificity in complex tissues requires systematic optimization:

• Antibody selection strategies:

  • Choose antibodies developed using computation-guided design approaches targeting unique epitopes

  • Select antibodies validated against multiple similar ligands to confirm specificity

  • Consider recombinant monoclonal antibodies which typically offer higher specificity than polyclonals

• Protocol optimization:

  • Implement stringent blocking steps using species-appropriate serum/BSA combinations

  • Optimize antibody concentration through careful titration experiments

  • Adjust incubation parameters (time, temperature, buffer composition)

  • Consider antigen retrieval modifications for fixed tissues

• Validation approaches:

  • Perform pre-adsorption experiments with blocking peptides specific to P2Y12

  • Include tissues with knockout/knockdown P2Y12 expression as negative controls

  • Confirm findings with alternative antibody clones targeting different epitopes

  • Correlate protein detection with mRNA expression data

• Advanced analytical methods:

  • Employ spectral imaging to distinguish specific signal from autofluorescence

  • Use computational image analysis to apply consistent signal thresholds

  • Consider single-cell analysis techniques to resolve heterogeneous expression

These approaches collectively enhance confidence in P2Y12 detection specificity, particularly important in tissues with complex cellular composition such as brain tissue .

What are the mechanisms underlying potential cross-reactivity of P2Y12 antibodies with other purinergic receptors?

Understanding potential cross-reactivity mechanisms is crucial for accurate P2Y12 detection:

• Structural similarity challenges:

  • P2Y12 shares significant sequence homology with other P2Y family members, particularly P2Y13 and P2Y14

  • Transmembrane domains are especially conserved across purinergic receptors

  • Extracellular loops, while more variable, may contain similar structural motifs

• Epitope-specific considerations:

  • Antibodies targeting the third extracellular loop (e.g., residues 270-282 in human P2Y12) show enhanced specificity

  • Antibodies against intracellular domains may cross-react with shared G-protein coupling motifs

  • Post-translational modifications can create or mask cross-reactive epitopes

• Experimental verification approaches:

  • Perform parallel staining with known P2Y1, P2Y13, and P2Y14 antibodies

  • Use heterologous expression systems to test antibodies against individual receptor subtypes

  • Consider computational epitope prediction to identify potential cross-reactive regions

  • Employ binding mode analysis to distinguish specific from non-specific interactions

• Emerging selection technologies:

  • Phage display selection against multiple ligands can identify antibodies with customized specificity profiles

  • High-throughput sequencing analysis helps identify binding modes associated with specific targets

  • Computational design approaches can optimize antibody sequences for improved discrimination

These considerations help researchers select and validate antibodies with minimal cross-reactivity to other purinergic receptors, improving experimental reliability .

How should researchers address weak signal issues when detecting P2Y12 in tissue samples?

When confronting weak P2Y12 signal intensity, implement this systematic troubleshooting approach:

• Sample preparation optimization:

  • Ensure proper tissue fixation (10% neutral buffered formalin for 24-48 hours typically optimal)

  • Optimize antigen retrieval method (Tris-EDTA buffer, pH 9.0, for 20 minutes recommended)

  • Consider section thickness (5-7μm typically provides optimal balance of signal and resolution)

• Antibody protocol enhancement:

  • Increase antibody concentration incrementally (test dilution series)

  • Extend primary antibody incubation time (overnight at 4°C may improve signal)

  • Try different detection systems (polymer-based versus avidin-biotin complex)

  • Employ signal amplification methods (tyramide signal amplification)

• Signal-to-noise optimization:

  • Implement more aggressive blocking of non-specific binding sites

  • Reduce background through additional washing steps

  • Minimize autofluorescence through appropriate quenching steps

  • Consider chromogenic versus fluorescent detection based on tissue type

• Instrumentation considerations:

  • Optimize microscope settings (exposure time, gain, offset)

  • Use appropriate filter sets for fluorescent detection

  • Consider spectral imaging to separate specific signal from autofluorescence

The optimal detection approach varies by tissue type and application context but should be systematically optimized and standardized for reliable results .

What factors should be considered when validating P2Y12 antibodies across different species?

Species validation of P2Y12 antibodies requires attention to several critical factors:

• Sequence homology analysis:

  • Human P2Y12 shares approximately 89% amino acid identity with mouse and 86% with rat

  • Epitope-specific sequence alignment should be performed before cross-species application

  • Focus on conservation in the specific region recognized by the antibody (e.g., residues 270-282 for extracellular antibodies)

• Validation approaches:

  • Perform side-by-side comparisons using identical protocols across species

  • Include positive control tissues for each species (brain tissue and platelets)

  • Compare staining patterns to published reference data for each species

  • Verify with orthogonal detection methods (mRNA expression, functional assays)

• Protocol adjustments:

  • Optimize antibody concentration independently for each species

  • Modify blocking solutions based on species-specific requirements

  • Adjust antigen retrieval conditions as needed for different tissue fixation methods

  • Select appropriate secondary antibodies specific to each primary antibody host species

• Data interpretation considerations:

  • Account for species differences in cellular distribution and expression levels

  • Consider developmental and regional expression variations across species

  • Document subtle differences in subcellular localization patterns

These methodical approaches ensure reliable cross-species application of P2Y12 antibodies, particularly important for translational research bridging animal models and human studies .

How can researchers quantitatively analyze P2Y12 expression in immunohistochemistry and flow cytometry?

Quantitative analysis of P2Y12 expression requires rigorous methodological approaches:

For Immunohistochemistry/Immunofluorescence:

• Image acquisition standardization:

  • Use consistent exposure settings across all experimental groups

  • Include calibration standards in each imaging session

  • Capture multiple representative fields per sample (minimum 5-10)

  • Implement blind coding of samples to prevent bias

• Analysis approaches:

  • Measure mean fluorescence intensity within defined cellular regions

  • Quantify percentage of P2Y12-positive cells within a population

  • Assess receptor distribution patterns (membrane versus cytoplasmic)

  • Apply threshold-based segmentation consistently across samples

• Software tools:

  • ImageJ/FIJI with appropriate plugins for batch processing

  • CellProfiler for automated cellular identification and quantification

  • HALO or QuPath for whole-slide tissue analysis

For Flow Cytometry:

• Assay standardization:

  • Use fluorescence calibration beads to standardize intensity measurements

  • Implement consistent gating strategies based on forward/side scatter and lineage markers

  • Apply the recommended antibody concentration (5 μL per million cells)

• Analysis parameters:

  • Mean/median fluorescence intensity (MFI) for expression level quantification

  • Percentage of positive cells using clearly defined positive thresholds

  • Population distribution analysis for heterogeneous samples

• Data presentation:

  • Report both percentage positive and MFI values

  • Present normalized data when comparing across experiments

  • Include appropriate statistical analysis for group comparisons

These approaches enable robust quantitative assessment of P2Y12 expression across experimental conditions and sample types .

How can P2Y12 antibodies be integrated into multiplex immunostaining protocols?

Incorporating P2Y12 antibodies into multiplex immunostaining requires strategic planning:

• Antibody panel design considerations:

  • Select antibodies from different host species to avoid cross-reactivity

  • Choose fluorophores with minimal spectral overlap

  • Consider signal intensity balancing (adjust antibody concentrations accordingly)

  • Plan marker combinations that answer specific biological questions

• Validated multiplex protocols:

  • Sequential staining approach: Apply, detect, and block/strip each primary antibody sequentially

  • Tyramide signal amplification: Enables use of multiple antibodies from same species

  • Spectral imaging: Permits separation of overlapping fluorophores through linear unmixing

• P2Y12-specific considerations:

  • Pair with cell-type specific markers (CD45, Iba1 for microglia; CD41 for platelets)

  • Include functional state markers (activation, maturation, polarization)

  • Consider subcellular colocalization studies with signaling pathway components

• Quality control measures:

  • Include single-stain controls for spectral unmixing

  • Perform fluorescence minus one (FMO) controls

  • Validate staining pattern consistency compared to single-marker staining

These approaches enable sophisticated analysis of P2Y12 in complex cellular contexts while maintaining detection specificity and sensitivity .

What are promising future directions for P2Y12 antibody development and application?

Emerging trends in P2Y12 antibody technology present exciting research opportunities:

• Next-generation antibody engineering:

  • Computational design of antibodies with customized specificity profiles

  • Development of conformational state-specific antibodies (active vs. inactive receptor)

  • Creation of bispecific antibodies targeting P2Y12 and complementary markers

  • Engineered antibodies with reduced immunogenicity for in vivo applications

• Novel detection technologies:

  • Super-resolution microscopy applications for nanoscale receptor distribution

  • Live-cell imaging with minimally disruptive antibody fragments

  • Mass cytometry (CyTOF) incorporation for high-dimensional analysis

  • Proximity ligation assays to study receptor-protein interactions

• Translational research applications:

  • Correlation of P2Y12 expression with clinical outcomes in cardiovascular disorders

  • Development of imaging agents for microglial visualization in neurological conditions

  • Monitoring of P2Y12 as a biomarker for antiplatelet therapy response

  • Investigation of P2Y12 in non-traditional cell types and pathologies

• Integration with -omics approaches:

  • Correlation of protein expression with transcriptomic and proteomic datasets

  • Single-cell analysis combining antibody detection with RNA sequencing

  • Systems biology approaches to understand P2Y12 regulatory networks

These directions represent promising frontiers in P2Y12 research, potentially yielding new insights into receptor biology and therapeutic applications .