CELSR3 Antibody, HRP conjugated

What is CELSR3 and why is it significant in neuroscience research?

CELSR3 belongs to the adhesion GPCR family that links extracellular adhesion to intracellular signaling. It plays a crucial role in interneuron development and function in the mammalian brain . CELSR3 is predominantly expressed in GABAergic inhibitory neurons across various forebrain regions including the olfactory bulb, cerebral cortex, hippocampus, and striatum . Its significance stems from its involvement in neural circuit formation and function, making it an important target for neurodevelopmental and neurodegenerative research.

How does CELSR3 differ structurally and functionally from other CELSR family members?

CELSR3 exhibits distinct structural and functional characteristics compared to its family members. Unlike CELSR2 which undergoes efficient autoproteolytic cleavage, CELSR1 and CELSR3 are cleavage deficient . This structural difference is likely due to variations in the GPS domain sequence. Despite these differences in autoproteolysis, all three CELSR proteins engage with GαS signaling pathways . Additionally, CELSR3 contains specific domains including cadherin repeats, EGF-like domains, and a seven-transmembrane region characteristic of GPCRs, which contribute to its unique functions in neuronal development and signaling.

What detection methods are suitable for CELSR3 protein analysis in neural tissues?

Several detection methods are appropriate for CELSR3 analysis in neural tissues:

• Immunohistochemistry (IHC): Particularly effective for paraffin-embedded or frozen sections of brain tissue to visualize regional expression patterns .

• Immunofluorescence (IF): Allows co-localization studies with other neuronal markers such as GABA to confirm expression in inhibitory neurons .

• Flow cytometry: Useful for quantitative analysis in neuronal cell populations, as demonstrated with SH-SY5Y human neuroblastoma cell lines .

• Western blotting: Appropriate for detecting full-length CELSR3 protein and confirming its non-cleaved status compared to other family members .

• ELISA: Provides quantitative measurement of CELSR3 levels in tissue homogenates or cell lysates .

For all methods, proper sample preparation and antibody optimization are essential for reliable results.

What are the key considerations when selecting a CELSR3 antibody for experimental applications?

When selecting a CELSR3 antibody, researchers should consider:

• Epitope specificity: Antibodies targeting different regions (N-terminal, middle domains, or C-terminal) may yield different results. For example, antibodies binding to AA 1601-1700 versus those targeting AA 531-711 may detect different populations or conformations of CELSR3 .

• Host species and clonality: Both polyclonal (offering broader epitope recognition) and monoclonal (providing consistent specificity) antibodies are available, with rabbit polyclonals and mouse monoclonals being common options .

• Cross-reactivity: Some CELSR3 antibodies cross-react with mouse, rat, and other species, while others are human-specific .

• Conjugate selection: HRP conjugation is advantageous for detection methods requiring enzymatic amplification, while fluorescent conjugates like PE are preferable for direct visualization or flow cytometry .

• Validation data: Review literature and supplier validation data for the specific detection methods you plan to employ.

How do different fixation and permeabilization protocols affect CELSR3 antibody binding and signal quality?

Fixation and permeabilization protocols significantly impact CELSR3 antibody binding due to its complex multi-domain structure and membrane localization:

For optimal results with CELSR3 antibodies, a sequential approach using 4% PFA followed by controlled permeabilization (0.1-0.3% Triton X-100) preserves the complex seven-transmembrane structure while allowing access to intracellular epitopes. Heat-induced epitope retrieval (citrate buffer, pH 6.0) may be necessary for formalin-fixed tissues to expose the epitope regions around AA 1601-1700 targeted by some antibodies .

What strategies can address potential cross-reactivity issues when studying CELSR3 in models expressing multiple CELSR family members?

When studying CELSR3 in systems expressing multiple CELSR family members, researchers should implement these strategies:

• Epitope mapping: Select antibodies targeting unique regions of CELSR3 not conserved in CELSR1/2. The region between AA 1601-1700 shows lower sequence homology compared to the highly conserved transmembrane domains .

• Validation controls:

  • Include CELSR3 knockout samples as negative controls

  • Pre-absorption with recombinant CELSR3 peptides to confirm specificity

  • Parallel staining with multiple antibodies targeting different CELSR3 epitopes

• Orthogonal approaches: Combine protein detection with mRNA analysis (in situ hybridization or RT-PCR) to confirm specificity of signals.

• Signal discrimination analysis: When using fluorescence-based detection, perform spectral unmixing to distinguish between closely related proteins.

• Selective inhibition: Use siRNA knockdown of specific CELSR family members to validate antibody specificity in complex tissues.

These approaches are particularly important when studying regions like the cerebral cortex where both CELSR2 and CELSR3 may be expressed .

How can researchers optimize western blot protocols for detecting full-length versus cleaved forms of CELSR3?

Optimizing western blot protocols for CELSR3 detection requires careful consideration of the protein's unique cleavage properties:

• Sample preparation:

  • Use mild lysis buffers (1% Triton X-100, 150mM NaCl, 50mM Tris pH 7.4) with protease inhibitor cocktail

  • Avoid repeated freeze-thaw cycles that may cause artifactual degradation

  • Include positive controls such as CELSR2 (efficiently cleaved) and LPHN3 for comparison

• Gel selection and running conditions:

  • Use 4-8% gradient gels for full-length CELSR3 (~350 kDa)

  • Lower percentage gels (6-8%) for potential N-terminal fragments

  • Extended running times at lower voltage (80-100V) improve resolution of high molecular weight proteins

• Transfer optimization:

  • Wet transfer with low SDS (0.01%) in transfer buffer

  • Extended transfer times (overnight at 30V, 4°C) for large proteins

  • Use PVDF membranes (0.45 μm pore size) for better protein retention

• Detection strategy:

  • Use antibodies targeting both N-terminal (HA-tagged) and C-terminal (FLAG-tagged) regions to differentiate between full-length and potential cleaved forms

  • Compare banding patterns with known cleavage-efficient aGPCRs like LPHN3

  • Include reducing and non-reducing conditions to assess potential disulfide-linked fragments

Research indicates that CELSR3 is predominantly detected as full-length protein (~350 kDa), with minimal cleavage products compared to CELSR2 .

What methodological approaches can distinguish between different signaling paradigms of CELSR3 beyond tethered agonist (TA) exposure?

To investigate CELSR3 signaling beyond the tethered agonist model, researchers can employ these methodological approaches:

• BRET2-based transducerome analysis:

  • Implement TRUPATH sensors for assaying multiple Gαβγ combinations (14 different combinations)

  • Combine with thrombin-mediated acute TA exposure systems to compare basal versus TA-dependent G protein coupling

  • Compare with PAR-fusion constructs to determine TA-independent signaling mechanisms

• Structure-function mutations:

  • Generate CELSR3 constructs with point mutations in the GPS domain

  • Create chimeric receptors by swapping domains between CELSR family members to identify regions critical for specific signaling pathways

  • Create TA point mutants to assess retention of GαS coupling activity

• Temporal signaling analysis:

  • Use rapid kinetic measurements with biosensors to detect immediate versus delayed signaling events

  • Apply specific G protein inhibitors (YM-254890 for Gq/11, PTX for Gi/o) to dissect pathway contributions

  • Implement optogenetic approaches for precise temporal control of receptor activation

• Biased signaling assessment:

  • Compare multiple downstream readouts (cAMP, IP1, ERK1/2 phosphorylation)

  • Use pathway-specific inhibitors to identify G protein-dependent versus arrestin-dependent signaling

  • Implement CRISPR-based knockout of specific signaling components

These approaches have revealed that CELSR3 exhibits both basal activity and can couple to GαS, with differential coupling efficacy compared to other CELSR family members .

What are the optimal substrate systems for HRP-conjugated CELSR3 antibody detection in different applications?

Optimal substrate systems for HRP-conjugated CELSR3 antibodies vary by application:

For CELSR3 detection in neural tissues where expression may be limited to specific neuronal populations, enhanced sensitivity substrates are recommended. When performing double-labeling experiments, select substrate combinations with distinct colors (e.g., DAB and Vector® Red) to clearly distinguish CELSR3 from other markers of GABAergic neurons .

How can signal amplification techniques be applied to enhance detection of low-abundance CELSR3 expression?

For detecting low-abundance CELSR3 expression, several signal amplification techniques can be employed:

• Tyramide Signal Amplification (TSA):

  • Provides 10-100 fold signal enhancement through catalyzed reporter deposition

  • Particularly useful for tissue sections with sparse CELSR3-expressing inhibitory neurons

  • Protocol adaptation: Use 1:1000-1:2000 HRP-conjugated CELSR3 antibody dilution with 5-minute tyramide incubation

• ABC (Avidin-Biotin Complex) method:

  • Use biotinylated secondary antibody followed by HRP-conjugated avidin-biotin complex

  • Particularly effective for fixed tissue sections where epitope accessibility may be limited

  • Combine with heat-mediated antigen retrieval for optimal results

• Polymer-based detection systems:

  • Employ dextran polymers conjugated with multiple HRP molecules and secondary antibodies

  • Reduces background while enhancing specific signal

  • Shorter protocol time compared to traditional ABC methods

• Sequential multiple antibody labeling:

  • Use cocktails of non-competing CELSR3 antibodies targeting different epitopes

  • Amplifies signal while confirming specificity through coincident detection

• Metal-enhanced DAB precipitation:

  • Incorporate nickel or cobalt salts into DAB substrate for intensified chromogenic signal

  • Provides superior contrast for morphological analysis of CELSR3-expressing neurons

Each method requires specific optimization for CELSR3 detection, with considerations for cellular compartmentalization given its membrane localization .

What are the potential artifacts and false positives when using HRP-conjugated CELSR3 antibodies, and how can they be mitigated?

Several artifacts can occur with HRP-conjugated CELSR3 antibodies:

• Endogenous peroxidase activity:

  • Origin: Particularly problematic in tissues rich in erythrocytes or myeloperoxidase-containing cells

  • Mitigation: Quench endogenous peroxidase with 0.3% H₂O₂ in methanol for 30 minutes before antibody incubation

  • Validation: Include primary antibody-omitted controls to identify non-specific signal

• Biotin-related background:

  • Origin: Endogenous biotin in tissues when using biotin-based detection systems

  • Mitigation: Pre-block with avidin/biotin blocking kit or use polymer-based detection systems

  • Validation: Compare biotin-based versus non-biotin detection methods

• Edge/trapping artifacts:

  • Origin: Non-specific binding at tissue edges or in necrotic regions

  • Mitigation: Optimize blocking (5% normal serum + 0.3% Triton X-100) and washing steps

  • Validation: Examine tissue morphology carefully in relation to signal distribution

• Cross-reactivity with related proteins:

  • Origin: Antibody recognition of other CELSR family members or related cadherins

  • Mitigation: Validate with CELSR3 knockout controls or peptide competition assays

  • Validation: Compare staining patterns with published CELSR3 expression in GABAergic neurons

• DAB precipitation artifacts:

  • Origin: Over-development leading to diffusion of chromogen beyond antigen sites

  • Mitigation: Monitor reaction development microscopically and optimize substrate incubation time

  • Validation: Compare with fluorescent detection methods for signal localization confirmation

How should researchers interpret differences in CELSR3 subcellular localization patterns across different neural cell types?

Interpretation of CELSR3 subcellular localization requires careful consideration of several factors:

• Neuronal type-specific distribution patterns:

  • In GABAergic interneurons: CELSR3 shows strong membrane localization with potential enrichment at synaptic contacts

  • In developing neurons: May show differential distribution in growth cones versus cell bodies

  • Analytical approach: Quantify membrane-to-cytoplasmic signal ratio across different neuronal populations identified by co-staining with cell-type markers

• Developmental stage considerations:

  • Early development: Potentially more diffuse cytoplasmic distribution related to ongoing trafficking

  • Mature neurons: More restricted to membrane compartments

  • Analysis method: Implement temporal cohort analysis with standardized imaging parameters

• Differential trafficking in healthy versus pathological conditions:

  • Normal conditions: Primarily surface localization as demonstrated in HEK293T cell studies

  • Stress conditions: May show internalization or altered compartmentalization

  • Quantification approach: Use line-scan analysis across cell boundaries to measure shifts in distribution

• Correlation with functional states:

  • Active signaling: Potential clustering in membrane microdomains

  • Receptor recycling: Increased vesicular compartmentalization

  • Analytical tool: Apply Manders' overlap coefficient for co-localization with trafficking markers

When analyzing subcellular localization, standardize image acquisition parameters and apply threshold-based segmentation for quantitative comparisons across experimental conditions .

What quantitative approaches can reconcile contradictory findings regarding CELSR3 expression levels across different experimental platforms?

To reconcile contradictory findings about CELSR3 expression across platforms:

• Multi-platform normalization strategy:

  • Implement parallel analysis of the same samples using at least three detection methods

  • Create conversion factors based on standard samples analyzed across platforms

  • Example approach: Normalize Western blot densitometry, qPCR, and immunofluorescence intensity using reference standards

• Absolute quantification methods:

  • Develop standard curves using recombinant CELSR3 protein fragments

  • Implement spike-in controls with known quantities of CELSR3 protein

  • Use digital PCR for absolute copy number determination at mRNA level

• Epitope accessibility analysis:

  • Compare antibodies targeting different domains (e.g., AA 1601-1700 versus AA 531-711)

  • Systematically evaluate effects of different sample preparation methods on detection

  • Correlate detection efficiency with protein structural predictions

• Statistical reconciliation approaches:

  • Meta-analysis techniques to integrate data from multiple studies

  • Weighted averaging based on methodological quality assessment

  • Bayesian integration of multiple measurement modalities

• Discrepancy investigation framework:

  • Decision tree for systematic evaluation of contradictory results

  • Analysis of potential biological versus technical variables

  • Controlled studies isolating specific methodological differences

These approaches help distinguish genuine biological variations from technical artifacts when comparing CELSR3 expression data across different experimental platforms.

How can researchers design experiments to distinguish between CELSR3 G-protein signaling versus potential G-protein-independent functions?

Experimental design to distinguish CELSR3 signaling mechanisms:

• CRISPR/Cas9-based signaling component knockouts:

  • Generate cell lines with selective knockout of specific G-protein subunits

  • Create CELSR3 mutants with impaired G-protein coupling but intact adhesion domains

  • Measure functional outcomes in parallel to assess contribution of each pathway

• Temporal dissection approach:

  • Compare rapid (seconds to minutes) versus delayed (hours) responses

  • Early responses typically reflect direct G-protein activation

  • Later responses may indicate G-protein-independent mechanisms

  • Implement time-series analysis with multiple downstream readouts

• Pharmacological dissection:

  • Apply G-protein inhibitors (e.g., YM-254890 for Gq/11, PTX for Gi/o)

  • Use BRET2-based sensors to monitor multiple G-protein coupling events simultaneously

  • Compare with known G-protein-dependent GPCR responses

• Adhesion versus signaling separation:

  • Develop truncation mutants separating adhesion domains from signaling domains

  • Create chimeric receptors fusing CELSR3 adhesion domains with non-GPCR cytoplasmic domains

  • Implement adhesion assays in parallel with signaling readouts

• Pathway-specific readouts:

  Signaling Pathway	Readout Method	Expected Timeframe	Controls
  GαS coupling	cAMP accumulation	5-30 minutes	Isoproterenol (β2-AR agonist)
  Gα13 pathway	SRE-luciferase	4-6 hours	Thrombin (PAR1 agonist)
  Adhesion function	Cell aggregation	30-60 minutes	Ca²⁺-free conditions
  Cytoskeletal effects	Neurite outgrowth	24-72 hours	Rho inhibitors

These experimental designs can help delineate the contribution of G-protein signaling versus potential G-protein-independent functions of CELSR3, similar to approaches used for LPHN3 .

How might multiplexed imaging approaches advance our understanding of CELSR3 expression in complex neural circuits?

Multiplexed imaging approaches offer powerful tools for understanding CELSR3 in neural circuits:

• Cyclic immunofluorescence (CycIF):

  • Sequential imaging of up to 30-40 proteins on the same tissue section

  • Application: Map CELSR3 expression relative to diverse neuronal and glial markers

  • Implementation: Include CELSR3 HRP-conjugated antibody in early cycles with tyramide-based amplification

  • Outcome: Comprehensive cellular context of CELSR3 expression across inhibitory neuron subtypes

• CODEX (CO-Detection by indEXing):

  • DNA-barcoded antibodies with iterative imaging

  • Application: High-parameter analysis of CELSR3 in spatial context

  • Implementation: Combine with oligonucleotide-tagged CELSR3 antibodies

  • Outcome: Quantitative analysis of CELSR3 distribution across brain regions

• Mass cytometry imaging (IMC):

  • Metal-tagged antibodies with laser ablation and mass spectrometry detection

  • Application: Quantitative mapping of CELSR3 and signaling components

  • Implementation: Metal-conjugated CELSR3 antibodies combined with phospho-specific signaling markers

  • Outcome: Spatial correlation of CELSR3 expression with signaling activity

• Expansion microscopy:

  • Physical tissue expansion for improved resolution

  • Application: Nanoscale distribution of CELSR3 in synaptic structures

  • Implementation: Post-expansion immunolabeling with HRP-conjugated antibodies

  • Outcome: Subcellular localization of CELSR3 at unprecedented resolution

• MERFISH (Multiplexed Error-Robust FISH):

  • Combining protein detection with multiplexed RNA visualization

  • Application: Correlate CELSR3 protein expression with transcript levels

  • Implementation: HRP-antibody detection followed by multiplexed RNA FISH

  • Outcome: Multi-scale analysis of CELSR3 regulation in neural circuits

These approaches can reveal how CELSR3 expression patterns correlate with functional neural circuit properties and may uncover previously unrecognized cell-type specificity .

What are the methodological challenges in developing conditional knockout systems for studying cell-type specific functions of CELSR3?

Developing conditional knockout systems for CELSR3 presents several methodological challenges:

• Gene targeting strategy optimization:

  • Challenge: CELSR3's large genomic locus (~356 kb) with 38 exons makes standard conditional allele design difficult

  • Solution: Target critical exons encoding the GPS domain or first transmembrane segment

  • Validation approach: Confirm complete protein loss using antibodies against multiple epitopes

• Cell-type specific Cre driver selection:

  • Challenge: Different interneuron subtypes may require distinct Cre lines for specific targeting

  • Solution: Implement intersectional genetic strategies (Cre/Flp) for improved specificity

  • Validation: Use reporter lines to confirm specificity before phenotypic analysis

• Temporal control implementation:

  • Challenge: Separating developmental versus mature functions of CELSR3

  • Solution: Use tamoxifen-inducible CreERT2 systems with tightly controlled induction protocols

  • Validation: Implement time-course analysis of protein depletion after induction

• Compensatory mechanism assessment:

  • Challenge: Potential upregulation of CELSR1/2 after CELSR3 deletion

  • Solution: Implement parallel RNA-seq and protein analysis for all family members

  • Validation: Compare acute versus chronic knockout phenotypes

• Functional readout selection:

  • Challenge: Identifying appropriate assays for CELSR3 function in specific cell types

  • Solution: Combine electrophysiological, morphological, and behavioral assessments

  • Validation: Compare with global knockout phenotypes where characterized

These methodological considerations are critical for accurately dissecting CELSR3 functions in specific neural populations while minimizing confounding factors from developmental compensation or incomplete targeting.