CAD8 Antibody

What is Cadherin-8 and what is its structural composition?

Cadherin-8 (CAD8/CDH8) belongs to the cadherin superfamily of cell adhesion molecules. Mature cadherin proteins feature a large N-terminal extracellular domain, a single membrane-spanning domain, and a small, highly conserved C-terminal cytoplasmic domain . In the nervous system, CAD8 protein signals are localized at synaptic clefts formed between central boutons (C-boutons) and dendritic processes organizing synaptic glomeruli, or in the vicinity of the synaptic cleft . Understanding this structure is crucial for selecting appropriate antibodies that target specific regions of the protein for different experimental applications.

What types of CAD8 antibodies are available for research applications?

Researchers can access various types of CAD8 antibodies including:

• Polyclonal antibodies targeting different regions (N-terminal, Middle region, Internal region)

• Monoclonal antibodies with specific epitope recognition

• Host options including rabbit and mouse-derived antibodies

• Conjugated and unconjugated formats for different detection methods
  When selecting an antibody, researchers should consider the specific epitope targeted, as antibodies directed against different regions of CAD8 may yield varying results depending on protein conformation and experimental conditions.

What are the typical applications for CAD8 antibodies in neuroscience research?

CAD8 antibodies are commonly employed in several experimental techniques:

• Western blotting for protein expression analysis

• Immunohistochemistry (IHC) on paraffin-embedded sections

• Immunofluorescence (IF) for cellular localization studies

• Enzyme immunoassays (EIA) for quantitative detection
  In neuroscience specifically, CAD8 antibodies have been valuable for characterizing synaptic structures and examining cell populations expressing this protein, such as in dorsal root ganglia (DRG) neurons and spinal cord interneurons .

How should sample preparation be optimized for CAD8 detection in neural tissues?

For optimal CAD8 detection in neural tissues, researchers should consider:

• Fixation method: Both 3.7% formaldehyde in PBS and 4% paraformaldehyde in calcium-containing buffers have been successful for tissue fixation .

• Calcium preservation: Avoid adding EDTA or EGTA in fixatives, as cadherins are easily degraded under calcium-free conditions .

• Tissue sectioning: For pupal or adult brain tissues, cutting samples into halves before fixation improves penetration and fixation quality .

• Epitope retrieval: May be necessary for paraffin-embedded tissues to expose the CAD8 epitope.
  When working with delicate neural structures, gentle processing is essential to maintain CAD8 localization at synaptic interfaces.

What controls should be included when using CAD8 antibodies in immunohistochemistry?

A robust experimental design for CAD8 immunohistochemistry should include:

• Positive control: Tissues known to express CAD8 (e.g., specific populations of DRG neurons)

• Negative control:

  • Primary antibody omission

  • Tissues from CAD8 knockout animals (such as cad8−/− mice)

  • Pre-adsorption with immunizing peptide

• Specificity control: Testing for cross-reactivity with related cadherins

• Method control: Alternative detection methods (e.g., in situ hybridization) to confirm protein expression patterns
  These controls help validate antibody specificity and differentiate true signals from background or non-specific binding.

How can researchers assess CAD8 antibody specificity?

Assessment of CAD8 antibody specificity can be approached through multiple methods:

• Western blot analysis: Compare wild-type and CAD8 knockout samples. In cad8−/− mice, no CAD8 protein should be detected with CAD8-specific antibodies .

• Comparative analysis: Test antibody reactivity against related cadherin family members to ensure no cross-reactivity.

• Epitope mapping: Confirm antibody binding to the intended region of CAD8.

• Immunoprecipitation followed by mass spectrometry: Verify that the antibody captures the intended protein.
  Documenting these specificity tests is crucial for publication and reproducibility of research findings.

How can CAD8 antibodies be utilized for studying synaptic organization in the nervous system?

CAD8 antibodies can provide valuable insights into synaptic organization through:

• Ultrastructural localization: Preembedding immunogold electron microscopy can precisely localize CAD8 at synaptic clefts, as demonstrated in studies of dorsal horn synaptic glomeruli .

• Co-localization studies: Double-immunolabeling with other synaptic markers helps identify specific synaptic subtypes expressing CAD8.

• Developmental profiling: Track CAD8 expression during synaptogenesis to understand its role in circuit formation.

• Activity-dependent changes: Investigate whether CAD8 distribution is altered following neuronal activation or in pathological conditions.
  These approaches help elucidate CAD8's role in organizing specific synaptic connections, particularly in sensory processing circuits.

What strategies can overcome technical challenges when using CAD8 antibodies in neural tissue?

Researchers face several technical challenges when working with CAD8 antibodies in neural tissue:

• Signal amplification: For low-abundance expression, consider using:

  • Tyramide signal amplification

  • Multiple-step secondary antibody detection

  • Highly sensitive detection systems

• Background reduction:

  • Extended blocking steps (2-3 hours) with serum matching the secondary antibody host

  • Addition of 0.1-0.3% Triton X-100 for improved antibody penetration

  • Careful titration of primary antibody concentration

• Tissue penetration:

  • For thick sections, extend incubation times (24-48 hours at 4°C)

  • Consider vibratome sections for better antibody access

• Preservation of morphology:

  • For ultrastructural studies, use specialized fixation protocols with calcium preservation

How can CAD8 antibodies be used to characterize cell populations in heterogeneous tissues?

For characterizing CAD8-expressing cell populations:

• Multi-color immunofluorescence: Combine CAD8 antibodies with markers for specific cell types. Previous studies have successfully combined CAD8/lacZ labeling with TRPM8 and TRPV1 to characterize sensory neuron subtypes .

• Flow cytometry: Use CAD8 antibodies for purification and quantification of specific cell populations.

• Single-cell analysis workflow:

  • Immunolabeling of CAD8-positive cells

  • FACS isolation of labeled populations

  • Single-cell RNA sequencing to determine molecular profiles

• Spatial transcriptomics: Combine CAD8 immunolabeling with in situ transcriptomics to correlate protein expression with gene expression patterns.
  This approach has revealed that approximately 76% of CAD8-positive sensory neurons express TRPM8, and 73% of TRPM8-positive neurons express CAD8, indicating a strong association between these markers .

How should researchers interpret conflicting results between different CAD8 antibodies?

When faced with conflicting results between different CAD8 antibodies:

• Compare epitope regions: Antibodies targeting different domains of CAD8 may yield different results due to:

  • Epitope accessibility in the native protein conformation

  • Post-translational modifications masking certain epitopes

  • Protein-protein interactions affecting antibody binding

• Validate with complementary techniques:

  • mRNA expression analysis (RT-PCR, in situ hybridization)

  • Genetic approaches (reporter gene expression in CAD8 locus)

  • Alternative antibodies targeting different epitopes

• Consider experimental conditions affecting epitope availability:

  • Fixation method

  • Antigen retrieval protocol

  • Detergent concentration

• Document all variables when reporting results, including antibody clone, lot number, and detailed methods.

What criteria should be used to validate CAD8 antibody specificity in knockout models?

When validating CAD8 antibodies using knockout models, researchers should apply these criteria:

• Complete absence of signal in CAD8 knockout tissues using Western blotting with antibodies specific for CAD8 protein .

• Consistent staining pattern between heterozygous and wild-type samples, with reduced intensity in heterozygous samples reflecting gene dosage.

• Correlation between immunohistochemical pattern and reporter gene expression (e.g., lacZ) when inserted into the CAD8 locus .

• Comparison of multiple antibodies targeting different epitopes to confirm consistent absence of signal.

• Verification that closely related cadherin family members still show normal expression patterns in the knockout, confirming specificity of the deletion.

How can researchers determine optimal antibody concentration for different applications?

Determining optimal antibody concentration requires systematic titration:

How do anti-CAD8 antibodies compare with genetic labeling approaches for studying CAD8-expressing cells?

Both antibody-based and genetic approaches offer complementary advantages:

• Antibody advantages:

  • Direct detection of endogenous protein

  • Applicable to wild-type tissues without genetic modification

  • Can detect post-translational modifications

  • Suitable for human samples

• Genetic reporter advantages:

  • Higher sensitivity for detecting low-expressing cells

  • Not dependent on protein stability or epitope accessibility

  • Allow for cell-type specific manipulation

  • Enable lineage tracing of CAD8-expressing populations

• Comparative considerations:

  • In CAD8 studies, lacZ reporter expression has been shown to mirror antibody staining patterns in most contexts

  • Reporter signal intensity may be stronger than antibody detection, particularly in heterozygous animals

  • Combined approaches (antibody detection in reporter lines) offer validation and enhanced sensitivity

What approaches can be used to study CAD8 protein interactions at synaptic interfaces?

To investigate CAD8 protein interactions at synapses:

• Proximity ligation assay (PLA):

  • Detects proteins within 40nm of each other

  • Visualizes potential CAD8 binding partners in situ

  • Provides spatial information about interaction sites

• Co-immunoprecipitation with synaptic preparations:

  • Isolate synaptic fractions (e.g., synaptosomes)

  • Immunoprecipitate CAD8 and identify binding partners

  • Validate interactions with reverse co-IP

• FRET/FLIM microscopy:

  • Requires fluorescently tagged proteins

  • Measures direct protein-protein interactions

  • Can be performed in living neurons

• Electron microscopy approaches:

  • Double immunogold labeling to visualize CAD8 with potential partners

  • Correlative light-electron microscopy for precise localization
    These methods can help elucidate how CAD8 contributes to synaptic organization and function in neural circuits.

How can CAD8 antibodies be used in combination with electrophysiology to correlate structure and function?

Integrating CAD8 immunolabeling with electrophysiology offers powerful insights:

• Post-recording immunohistochemistry workflow:

  • Perform patch-clamp recordings from neurons

  • Fill cells with biocytin or fluorescent dye

  • Process tissue for CAD8 immunolabeling

  • Correlate CAD8 expression with recorded electrophysiological properties

• Antibody-based manipulation during recordings:

  • Apply CAD8 function-blocking antibodies during electrophysiological recording

  • Monitor changes in synaptic transmission

  • Assess specific effects on synapses containing CAD8

• Optogenetic targeting of CAD8-expressing neurons:

  • Use CAD8 promoter-driven expression of channelrhodopsin

  • Combine with CAD8 immunolabeling to confirm targeting specificity

  • Record light-evoked responses in connected neurons These combined approaches can reveal how CAD8-mediated adhesion influences synaptic transmission and circuit function in the nervous system.