CELSR3 Antibody, FITC conjugated

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

CELSR3 (Cadherin EGF LAG Seven-Pass G-Type Receptor 3) is an atypical cadherin functioning as an adhesion G protein-coupled receptor (aGPCR) that plays critical roles in brain development. It is expressed highly in postnatal Purkinje cells and is essential for proper neuronal development and function. Studies with conditional knockout mice demonstrate that CELSR3 is required for Purkinje cell maturation and regulates postsynaptic plasticity in the cerebellum . Unlike many other aGPCRs including related family members, CELSR3 displays an unusual GAIN domain where the typical catalytic threonine/serine cleavage residue is replaced with glycine, resulting in predominantly uncleaved, full-length receptors . This structural distinction makes CELSR3 a particularly interesting target for developmental neurobiology and synaptic plasticity studies.

What epitope does the CELSR3 Antibody, FITC conjugated recognize?

The CELSR3 Antibody, FITC conjugated (product code CSB-PA852817LC01HU) is raised against a recombinant human CELSR3 protein fragment spanning amino acids 514-718 . This region is part of the extracellular domain of the protein. The antibody is developed in rabbit as a polyclonal IgG and purified using Protein G chromatography to achieve >95% purity . The specificity for this particular epitope allows researchers to detect CELSR3 in its native conformation in human samples.

What is the difference between CELSR3 and other CELSR family members?

CELSR3 differs from its family members CELSR1 and CELSR2 in several key aspects:

This distinction in protein processing is significant for functional studies, as CELSR3 and CELSR1 remain predominantly as full-length receptors, while CELSR2 undergoes autoproteolytic cleavage . Functionally, CELSR3 is particularly important in neuronal development and has been shown to be critical for motor coordination and synaptic plasticity in the cerebellum .

What are the validated applications for CELSR3 Antibody, FITC conjugated?

The FITC-conjugated CELSR3 antibody has been validated for several immunological applications, allowing direct fluorescent detection without secondary antibodies. Based on available data, the antibody can be used in:

• Immunofluorescence (IF) - allowing direct visualization of CELSR3 expression patterns in cells and tissues

• ELISA - for quantitative detection of CELSR3 in biological samples

For broader application range, other CELSR3 antibodies have been validated for:

• Immunohistochemistry with paraffin-embedded sections (IHC-P)

• Immunohistochemistry with frozen sections (IHC-F)

• Immunocytochemistry (ICC)

• Western blotting (WB)

Recommended working dilutions for various applications are:

• ELISA: 1:500-1000

• IF: 1:50-200

• IHC-P: 1:200-400

• IHC-F: 1:100-500

• ICC: 1:100-500

How should I design experiments to visualize CELSR3 in neuronal tissue sections?

For visualizing CELSR3 in neuronal tissues, particularly in the cerebellum where CELSR3 shows high expression in Purkinje cells, consider the following experimental approach:

• Tissue preparation: For optimal results with CELSR3 antibodies, both fresh-frozen and paraffin-embedded sections can be used. In studies of postnatal cerebellar development, tissues from different developmental stages (P3, P10, etc.) provide valuable insights into temporal expression patterns .

• Dual-labeling strategy: Combine CELSR3 Antibody, FITC conjugated with antibodies against neuronal markers such as Calbindin (Purkinje cell marker) using a compatible fluorophore. This approach has successfully demonstrated that all Calbindin-positive Purkinje cells express CELSR3-GFP in mouse models .

• Confocal microscopy: Use confocal microscopy to capture high-resolution images that allow visualization of CELSR3 localization in Purkinje cell bodies and dendrites.

• Controls: Include appropriate negative controls (omitting primary antibody) and positive controls (tissues known to express CELSR3) to validate specificity.

• Quantification: For quantitative analysis, measure fluorescence intensity across developmental stages or experimental conditions to track changes in CELSR3 expression.

Research demonstrates that CELSR3 expression can be observed in Purkinje cell bodies and their dendritic arbors in the molecular layer of the cerebellum , making these structures excellent targets for immunofluorescence studies.

What are the optimal storage and handling conditions for CELSR3 Antibody, FITC conjugated?

To maintain the integrity and activity of CELSR3 Antibody, FITC conjugated:

• Storage temperature: Upon receipt, store at -20°C or -80°C. The antibody is provided in a storage buffer containing 50% glycerol, 0.01M PBS (pH 7.4), and 0.03% Proclin 300 as a preservative .

• Freeze-thaw cycles: Avoid repeated freeze-thaw cycles as these can degrade the antibody and diminish the fluorescent signal from the FITC conjugate .

• Working solution preparation: When preparing working dilutions, use sterile techniques and buffer solutions free of contamination.

• Light sensitivity: As with all FITC-conjugated antibodies, protect from prolonged exposure to light to prevent photobleaching of the fluorophore.

• Stability: When stored properly, the antibody typically remains stable for at least 12 months from the date of receipt .

• Safety considerations: Note that the preservative Proclin 300 in the storage buffer is classified as poisonous and hazardous, requiring handling by trained personnel .

How can I optimize staining protocols to improve signal-to-noise ratio with this antibody?

To achieve optimal signal-to-noise ratio when working with CELSR3 Antibody, FITC conjugated:

• Antigen retrieval: For fixed tissue sections, especially paraffin-embedded specimens, appropriate antigen retrieval methods (heat-induced or enzymatic) may be necessary to expose epitopes masked during fixation.

• Blocking optimization: Use 1-5% BSA or normal serum from a species different from the host species of the primary antibody (not rabbit) to reduce non-specific binding.

• Antibody titration: Perform a titration experiment using different dilutions (starting with manufacturer's recommendations of 1:50-200 for IF applications) to determine the optimal concentration that provides specific signal with minimal background .

• Washing steps: Include sufficient washing steps (at least 3×5 minutes) with PBS containing 0.05-0.1% Tween-20 to remove unbound antibody.

• Counterstains: Select nuclear counterstains compatible with FITC fluorescence, such as DAPI, which emits in the blue spectrum and doesn't interfere with the green FITC signal.

• Autofluorescence reduction: For tissues with high autofluorescence (especially brain tissue), consider treatments with Sudan Black B or commercial autofluorescence reducers prior to immunostaining.

• Mounting medium: Use anti-fade mounting medium specifically formulated for fluorescence to prevent photobleaching during analysis.

How can I use CELSR3 Antibody to investigate synaptic plasticity mechanisms?

CELSR3 has been implicated in synaptic plasticity, particularly in long-term potentiation (LTP) in Purkinje cells. To investigate these mechanisms:

• Electrophysiology with immunofluorescence: Combine electrophysiological recording techniques with post-hoc immunofluorescence staining using CELSR3 Antibody, FITC conjugated. This approach allows correlation between CELSR3 expression levels and functional synaptic properties in the same cells.

• Synapse quantification: Use the antibody in conjunction with synaptic markers to evaluate the impact of CELSR3 on synapse formation and maintenance. Research has shown that CELSR3 conditional knockout mice exhibit decreased synaptic density in cerebellar tissue (7.9 ± 0.85/100 μm² compared to 13.7 ± 0.96/100 μm² in controls) .

• LTP/LTD analysis: In studies examining the role of CELSR3 in LTP and long-term depression (LTD), the antibody can be used to verify CELSR3 expression in parallel with electrophysiological recordings. Research indicates that CELSR3 is required for Wnt5a-induced enhancement of LTP, which is mediated through cAMP signaling .

• Co-localization studies: Investigate the interaction between CELSR3 and other components of the Wnt signaling pathway by performing co-localization studies with antibodies against Wnt5a, Frizzled receptors, or downstream effectors.

• Subcellular localization: Examine the distribution of CELSR3 in dendritic spines and postsynaptic densities using high-resolution imaging techniques such as super-resolution microscopy or electron microscopy with immunogold labeling.

What approaches can be used to study CELSR3 interactions with G proteins using this antibody?

CELSR3 functions as an adhesion G protein-coupled receptor (aGPCR), interacting with G proteins to mediate signal transduction. To investigate these interactions:

• Co-immunoprecipitation: Use CELSR3 Antibody to pull down CELSR3 complexes and analyze co-precipitated G proteins. This approach can identify which G protein subunits interact with CELSR3 under various conditions.

• Proximity ligation assay (PLA): Combine CELSR3 Antibody, FITC conjugated with antibodies against specific G protein subunits in a PLA to visualize direct interactions in situ with subcellular resolution.

• FRET/BRET analysis: For live-cell studies, complementary approaches using fluorescence or bioluminescence resonance energy transfer can detect CELSR3-G protein interactions in real-time.

• G protein coupling assays: Integrate antibody detection with functional assays that measure G protein activation, such as GTPγS binding or BRET-based G protein dissociation assays.

Research has demonstrated that CELSR3, unlike some other aGPCRs, remains predominantly as an uncleaved, full-length receptor due to its unique GAIN domain structure . This characteristic may influence its G protein coupling mechanisms, potentially distinguishing it from other family members that undergo autoproteolytic cleavage.

What are common problems encountered when using FITC-conjugated antibodies in neuronal tissue and their solutions?

When working with CELSR3 Antibody, FITC conjugated in neuronal tissues, researchers may encounter several challenges:

• High background/autofluorescence:

  • Problem: Brain tissue naturally contains lipofuscin and other autofluorescent components.

  • Solution: Pretreat sections with Sudan Black B (0.1-0.3% in 70% ethanol) for 10-20 minutes after immunostaining but before mounting. Alternatively, use specialized autofluorescence quenching reagents or imaging techniques that can distinguish between autofluorescence and specific signal.

• Weak or absent signal:

  • Problem: Insufficient antibody penetration or epitope masking.

  • Solution: Optimize fixation time, test different antigen retrieval methods, increase antibody concentration, or extend incubation time (overnight at 4°C). For thick sections, consider using detergents like Triton X-100 (0.1-0.3%) to improve antibody penetration.

• Photobleaching:

  • Problem: FITC is relatively susceptible to photobleaching.

  • Solution: Minimize exposure to light during staining and microscopy. Use anti-fade mounting media containing protective agents like p-phenylenediamine or proprietary anti-fade compounds. Consider acquiring images from unexposed fields first.

• Cross-reactivity:

  • Problem: Non-specific binding to other proteins.

  • Solution: Increase blocking time/concentration and perform additional validation using CELSR3 knockout tissues as negative controls or peptide competition assays.

• Inconsistent staining patterns:

  • Problem: Variable expression levels or processing artifacts.

  • Solution: Standardize tissue collection, fixation protocols, and staining procedures. Include internal positive controls in each experiment.

How can I validate the specificity of observed CELSR3 staining patterns?

• Genetic controls: The gold standard for antibody validation is comparing staining patterns between wild-type and knockout tissues. Conditional CELSR3 knockout mouse models have been developed and can serve as excellent negative controls .

• Peptide competition: Pre-incubate the antibody with excess immunizing peptide (CELSR3 fragment AA 514-718) before staining to block specific binding sites.

• Multiple antibodies approach: Compare staining patterns using different antibodies targeting distinct epitopes of CELSR3. Consistent patterns across antibodies increase confidence in specificity.

• Correlation with mRNA expression: Perform in situ hybridization for CELSR3 mRNA and compare with protein expression patterns detected by immunofluorescence.

• Western blot validation: Confirm antibody specificity by Western blot analysis of tissue lysates, looking for bands of the expected molecular weight (~358 kDa for full-length CELSR3).

• Developmental or experimental validation: CELSR3 expression changes during development, with high expression in postnatal Purkinje cells . Confirming these established patterns provides another validation method.

• Co-localization with known markers: Verify that CELSR3 staining co-localizes with established markers in expected cell types, such as Calbindin in Purkinje cells .

How does CELSR3 function differ between developmental stages and in pathological conditions?

CELSR3 exhibits distinct functions across developmental stages and can be dysregulated in pathological conditions:

• Developmental roles:

  • Embryonic development: CELSR3 is critical for brain embryonic development, particularly in neuronal migration and axon guidance .

  • Postnatal development: In the postnatal cerebellum, CELSR3 shows high expression in Purkinje cells and is required for their proper maturation .

  • Maturation phase: CELSR3 regulates dendritic arborization and spine formation in maturing neurons, with conditional knockout mice exhibiting atrophic Purkinje cell dendrites and decreased synapse density .

• Adult brain function:

  • Synaptic plasticity: CELSR3 is involved in maintaining synaptic connections and regulating synaptic plasticity, including both long-term potentiation (LTP) and long-term depression (LTD) .

  • Motor coordination: Mice with conditional knockout of CELSR3 in Purkinje cells show deficits in motor coordination and learning, highlighting its ongoing importance in adult cerebellar function .

• Pathological implications:

  • Neurodevelopmental disorders: Given its role in neuronal development, CELSR3 dysfunction may contribute to neurodevelopmental disorders affecting motor coordination and learning.

  • Synaptic dysfunction: The reduction in synaptic density (approximately 42% decrease) and alterations in synaptic transmission (reduced mEPSC frequency) observed in CELSR3 conditional knockout mice suggest potential implications for conditions involving synaptic pathology .

Investigating these stage-specific and context-dependent functions requires careful experimental design, with appropriate time points and models to capture the dynamic roles of CELSR3.

What is known about the molecular mechanisms of CELSR3 signaling in neuronal circuits?

CELSR3 mediates signaling through several molecular mechanisms in neuronal circuits:

• Wnt signaling pathway integration:

  • CELSR3 functions in non-canonical Wnt signaling pathways, particularly interacting with Wnt5a

  • Wnt5a perfusion enhances LTP formation in cerebellar slices, but this effect is abolished in CELSR3 conditional knockout mice

  • This Wnt5a-induced enhancement of LTP is mediated through cAMP signaling, as it can be occluded by cAMP agonists and diminished by cAMP antagonists

• G protein coupling mechanisms:

  • As an adhesion G protein-coupled receptor (aGPCR), CELSR3 can engage G proteins to transduce signals

  • Unlike many aGPCRs that undergo autoproteolytic cleavage exposing a tethered agonist, CELSR3 predominantly exists as an uncleaved receptor due to the replacement of the catalytic threonine/serine with glycine in its GAIN domain

  • This structural distinction suggests potentially unique G protein coupling mechanisms compared to other aGPCRs

• Synaptic organization:

  • CELSR3 regulates the density of asymmetrical synapses on Purkinje cell dendrites, with approximately 42% fewer synapses observed in conditional knockout mice

  • While affecting synapse number, CELSR3 does not appear to influence the thickness of postsynaptic densities

  • CELSR3 conditional knockout leads to reduced frequency (but not amplitude) of miniature excitatory postsynaptic currents (mEPSCs), indicating a role in presynaptic release probability or synapse number rather than postsynaptic receptor content

• Postsynaptic plasticity regulation:

  • CELSR3 is required for both LTP and LTD at parallel fiber-Purkinje cell synapses

  • The cAMP signaling pathway can still induce LTP independently of CELSR3, suggesting that CELSR3 functions upstream of cAMP in this signaling cascade

Understanding these molecular mechanisms can guide experimental approaches using CELSR3 antibodies to investigate specific signaling components and their relationships in neuronal development and function.

How might CELSR3 antibodies be used in emerging brain organoid and 3D culture systems?

CELSR3 antibodies, including FITC-conjugated variants, offer valuable tools for investigating neurodevelopmental processes in advanced 3D culture systems:

• Developmental trajectory mapping:

  • Brain organoids recapitulate many aspects of human brain development, making them ideal for studying CELSR3's role in neuronal migration and circuit formation

  • CELSR3 antibodies can track expression patterns across developmental stages in organoids, correlating with morphological and functional changes

  • Time-course immunostaining with CELSR3 Antibody, FITC conjugated would allow direct visualization of protein expression without secondary antibody complications in the complex 3D environment

• Comparison of normal and pathological development:

  • Organoids derived from patient iPSCs with neurodevelopmental disorders can be compared with control organoids

  • CELSR3 immunostaining patterns may reveal differences in expression or localization associated with pathological conditions

  • Quantitative analysis of CELSR3 distribution relative to cellular organization might identify subtle defects in neuronal positioning or connectivity

• Live imaging applications:

  • For live organoid imaging, membrane-permeant CELSR3 antibody fragments conjugated with FITC could potentially track receptor dynamics in real-time

  • This approach would require careful validation but could provide unprecedented insights into CELSR3 trafficking and localization during active developmental processes

• Functional correlation studies:

  • Combining CELSR3 immunostaining with calcium imaging or electrophysiological recordings from organoids would connect molecular expression patterns with functional circuit development

  • This multimodal approach could reveal how CELSR3 expression correlates with the emergence of synchronized network activity

What are the technical considerations for using CELSR3 antibodies in super-resolution microscopy studies?

Super-resolution microscopy techniques offer opportunities to study CELSR3 localization and interactions at nanoscale resolution, with several important considerations:

• FITC compatibility with super-resolution techniques:

  • FITC is compatible with STED (Stimulated Emission Depletion) microscopy, though its photostability is limited

  • For STORM/PALM techniques, FITC is suboptimal due to its relatively poor photoswitching properties

  • Consider using the unconjugated CELSR3 antibody with secondary antibodies linked to dyes optimized for super-resolution (e.g., Alexa Fluor 647 for STORM)

• Sample preparation optimization:

  • Super-resolution requires exceptional sample quality with minimized background

  • For neuronal tissues, optimize fixation protocols (4% PFA for 10-20 minutes is often optimal)

  • Consider using thinner sections (10-20 μm) to reduce out-of-focus background

  • Include additional blocking steps and extended washing to minimize non-specific binding

• Multi-color imaging strategies:

  • For co-localization studies of CELSR3 with synaptic proteins at nanoscale resolution

  • Select fluorophore combinations with minimal spectral overlap and compatible for the chosen super-resolution technique

  • Consider sequential immunostaining protocols to minimize antibody cross-reactivity when multiple primary antibodies from the same species are used

• Quantitative analysis approaches:

  • Develop analysis workflows to quantify nanoscale distribution patterns of CELSR3

  • Measure clustering parameters, nearest-neighbor distances, or co-localization with synaptic markers at super-resolution

  • Compare these parameters between experimental conditions (e.g., wild-type vs. CELSR3 conditional knockout samples)

• Validation controls:

  • Include rigorous controls specific to super-resolution microscopy

  • Single-color controls to measure and correct for spectral bleed-through

  • Primary antibody omission controls to assess non-specific binding of secondary antibodies

  • Known biological structures as positive controls to validate resolution and staining quality