CCD7 Antibody

What is the CCD7-1 antibody and what does it target?

CCD7-1 is a monoclonal antibody that specifically targets cadherin-7 (CDH7), a calcium-dependent cell adhesion protein. It was generated using recombinant chicken cadherin-7 (N-terminal 597 amino acids) fused to human Fc and expressed in COS-7 cells as the immunogen . The antibody recognizes the extracellular domain of cadherin-7 and has been epitope-mapped. CCD7-1 was deposited to the Developmental Studies Hybridoma Bank (DSHB) by researchers Takeichi and Nakagawa from Kyoto University in 2003 .

What are the validated experimental applications for CCD7-1 antibody?

CCD7-1 antibody has been validated for multiple research applications including:

• Fluorescence-activated cell sorting (FACS)

• Immunofluorescence microscopy

• Immunohistochemistry

• Immunoprecipitation

• Western blotting

Each application requires specific optimization protocols, with Western blotting typically requiring dilution determination for detecting the target protein, which has a predicted molecular weight of 82 kDa but appears at approximately 105 kDa on gels (with additional lower molecular weight bands) .

What species reactivity has been confirmed for CCD7-1 antibody?

The CCD7-1 antibody has confirmed reactivity across several vertebrate species:

• Chicken (Gallus gallus)

• Quail

• Turtle

• Lizard

This cross-species reactivity makes it valuable for comparative studies across vertebrate lineages, particularly in developmental biology and neuroscience research examining evolutionary conservation of cadherin-7 function.

How should CCD7-1 antibody be stored and handled to maintain optimal activity?

For short-term use (up to two weeks), CCD7-1 can be stored at 4°C. For long-term storage, divide the antibody solution into small aliquots (minimum 20 μl) and store at -20°C or -80°C to prevent activity loss through freeze-thaw cycles . When using frozen aliquots, thaw completely at room temperature before use and centrifuge briefly to collect the solution at the bottom of the tube. For concentrate or bioreactor products, equal volumes of glycerol can be added for storage stability .

What controls should be included when using CCD7-1 antibody in immunohistochemistry?

When designing immunohistochemistry experiments with CCD7-1:

• Positive control: Include tissue sections known to express cadherin-7 (e.g., specific neural tissues in chicken embryos)

• Negative control: Include sections from tissues where cadherin-7 is not expressed or knockdown models

• Isotype control: Include matched mouse IgG1 with kappa light chain (the isotype of CCD7-1)

• Secondary antibody-only control: Omit primary antibody to assess nonspecific binding

These controls allow for accurate assessment of specific staining versus background and are essential for publication-quality research.

How can blocking be optimized when performing immunofluorescence with CCD7-1 antibody?

Optimizing blocking conditions is crucial for reducing background and improving signal-to-noise ratio when using CCD7-1 antibody. Based on antibody characteristics:

• Use 5-10% normal serum (from the species in which the secondary antibody was raised) in PBS with 0.1-0.3% Triton X-100 for permeabilization

• Include 1-2% BSA to reduce nonspecific protein interactions

• Consider adding 0.1% cold fish skin gelatin to further reduce background

• Block for 1-2 hours at room temperature

• For tissues with high endogenous biotin, include an avidin-biotin blocking step if using biotin-based detection systems

If background persists, incorporate a pre-adsorption step with the relevant species tissues or increase blocking agent concentration.

How can CCD7-1 be adapted for use in flow cytometry for rare cell population detection?

For detecting rare cadherin-7 expressing cell populations using CCD7-1 in flow cytometry:

• Titrate antibody concentration (typically starting at 1-10 μg/ml) to determine optimal signal-to-noise ratio

• Use indirect staining with fluorophore-conjugated secondary antibodies for signal amplification

• Include a viability dye to exclude dead cells that can bind antibodies nonspecifically

• Implement a pre-enrichment step using magnetic beads if target population is <1%

• Employ multi-parameter gating strategies incorporating additional markers to identify the cadherin-7+ population within specific lineages

• Consider using PBS with 0.5% BSA and 2mM EDTA as staining buffer to prevent calcium-dependent homophilic binding of cadherins during processing

This approach enables precise quantification of cadherin-7 expression in developmental or pathological contexts.

What are the key considerations when using CCD7-1 for co-immunoprecipitation of cadherin-7 binding partners?

When using CCD7-1 for co-immunoprecipitation studies:

• Lysis buffer selection is critical - use buffers containing 1% NP-40 or similar mild non-ionic detergents that preserve protein-protein interactions

• Include calcium (1-2 mM CaCl₂) in all buffers as cadherin interactions are calcium-dependent

• Pre-clear lysates with protein G beads to reduce nonspecific binding

• Cross-link the antibody to beads using dimethyl pimelimidate (DMP) to prevent antibody co-elution

• Consider native elution conditions using competitive peptides rather than denaturing conditions if maintaining complex integrity is important

• Validate results with reverse co-immunoprecipitation using antibodies against suspected binding partners

• Confirm antibody specificity with appropriate knockdown controls

This methodological approach enables identification of novel cadherin-7 interaction partners in different developmental and cellular contexts.

How should researchers address epitope masking issues when using CCD7-1 antibody?

Epitope masking can occur when the CCD7-1 epitope in the extracellular domain of cadherin-7 is obscured by protein interactions, conformational changes, or fixation artifacts. To address this issue:

• Compare multiple fixation protocols (4% PFA, methanol, acetone) to determine optimal epitope preservation

• Implement antigen retrieval methods:

  • Heat-induced epitope retrieval: citrate buffer (pH 6.0) or Tris-EDTA (pH 9.0)

  • Enzymatic retrieval: light protease treatment (0.01-0.1% trypsin) for 5-10 minutes

• Test different detergent permeabilization conditions (0.1-0.5% Triton X-100 or 0.1-0.5% Saponin)

• Consider native versus denatured conditions for applications like Western blotting

• If complexed with other cadherins, include calcium chelators (5mM EDTA) in pre-treatment steps to disrupt cadherin interactions

These approaches help maximize detection sensitivity when epitope accessibility is compromised by biological or technical factors.

How does CCD7-1 antibody performance compare with anti-CD7 antibodies used in T-cell research?

It's essential to distinguish between CCD7-1 (targeting cadherin-7) and anti-CD7 antibodies (targeting the CD7 T-cell surface protein), as confusion between these can lead to experimental misinterpretation:

While both are monoclonal antibodies, they recognize entirely different proteins with distinct biological functions and research applications . The significantly different molecular weights (105 kDa vs. 40 kDa) provide a clear way to verify correct target detection in Western blot applications.

What considerations are important when analyzing CCD7-1 staining patterns in developmental tissue samples?

When interpreting CCD7-1 staining in developmental contexts:

• Temporal specificity: Cadherin-7 expression changes during development, requiring precise staging of samples

• Spatial heterogeneity: Expression may be restricted to specific tissues, requiring careful anatomical orientation and sectioning

• Subcellular localization: Distinguish between membrane-localized (functional) and cytoplasmic (potentially trafficking) cadherin-7

• Cell-type specificity: Use co-staining with lineage markers to identify exactly which cells express cadherin-7

• Intensity quantification: Implement standardized exposure settings and quantitative image analysis for comparing expression levels

• Developmental dynamics: Serial sections or time-course studies may be necessary to capture transient expression patterns

When publishing, include comprehensive documentation of developmental stage, precise anatomical regions, and clear demarcation of expression boundaries .

How should researchers differentiate between specific and non-specific binding when using CCD7-1 in cross-species applications?

When applying CCD7-1 antibody across different species:

• Sequence homology analysis: Compare the epitope region sequence of chicken cadherin-7 with that of the target species to predict likely cross-reactivity

• Validation approaches:

  • Western blot analysis showing the expected molecular weight (accounting for species variation)

  • Absorption controls using recombinant cadherin-7 from the species being studied

  • Knockdown or knockout validation if possible in the target species

• Multiple detection methods: Confirm results using at least two different techniques (e.g., immunohistochemistry and Western blotting)

• Comparison with RNA expression data: Correlate protein detection with mRNA expression patterns from in situ hybridization or transcriptomics

• Conservative interpretation: Acknowledge limitations and potential cross-reactivity with other cadherin family members in the discussion of results

This methodical approach ensures reliable interpretation of cross-species immunolabeling studies.

How can CCD7-1 antibody be used to study cadherin-7's role in neural development?

To investigate cadherin-7's functions in neural development using CCD7-1:

• Spatiotemporal mapping: Perform whole-mount immunostaining or section immunohistochemistry throughout developmental stages to create an expression atlas

• Circuit tracing: Combine with neuronal tracers to correlate cadherin-7 expression with specific neural circuits

• Ex vivo functional analysis:

  • Apply CCD7-1 to explant cultures to block cadherin-7 function

  • Observe effects on neurite outgrowth, fasciculation, or synaptogenesis

• In vivo perturbation: Combine with in ovo electroporation of dominant-negative constructs and use CCD7-1 to verify expression patterns

• Synaptic localization: Perform double-immunolabeling with synaptic markers to assess enrichment at specific synapse types

• Activity dependence: Examine changes in cadherin-7 expression following neural activity manipulation and detect with CCD7-1

These approaches help delineate cadherin-7's specific roles in neural circuit formation and maintenance.

What modifications are necessary when using CCD7-1 antibody for electron microscopy studies?

For successful immunoelectron microscopy with CCD7-1:

• Fixation optimization:

  • Use light fixation (0.5-2% paraformaldehyde with or without 0.1-0.5% glutaraldehyde)

  • Shorten fixation time to preserve antigenicity while maintaining ultrastructure

• Embedding considerations:

  • For pre-embedding: Perform immunolabeling before embedding in resin

  • For post-embedding: Use acrylic resins (e.g., LR White or Lowicryl) rather than epoxy resins

• Signal enhancement:

  • Utilize gold-conjugated secondary antibodies (typically 5-15nm particles)

  • Consider silver enhancement for improved visibility

• Permeabilization balance:

  • Use lower detergent concentrations (0.05-0.1% saponin) to maintain ultrastructure

  • Apply detergent in controlled, brief exposures

• Background reduction:

  • Pre-adsorb antibodies against fixed tissue components

  • Use smaller gold particles for better penetration and lower steric hindrance

• Controls:

  • Include immunogold labeling with isotype control at identical concentration

  • Perform peptide competition controls to verify specificity

These modifications enable subcellular localization of cadherin-7 at the ultrastructural level while preserving epitope accessibility.

What strategies can resolve weak or absent CCD7-1 antibody signal in Western blotting?

When troubleshooting weak or absent CCD7-1 signal in Western blots:

• Sample preparation:

  • Ensure complete solubilization of membrane proteins using appropriate detergents (1% Triton X-100 or 0.5% SDS)

  • Include protease inhibitors to prevent cadherin-7 degradation

  • Maintain samples at 4°C during processing

• Loading and transfer:

  • Increase protein loading (50-100 μg total protein per lane)

  • Optimize transfer conditions for high molecular weight proteins (105 kDa)

  • Use PVDF membrane instead of nitrocellulose for better protein retention

• Antibody incubation:

  • Increase primary antibody concentration (5-10 μg/ml)

  • Extend incubation time (overnight at 4°C)

  • Use 5% non-fat dry milk in TBST as blocking buffer

• Detection enhancement:

  • Implement signal amplification systems (biotin-streptavidin)

  • Increase exposure time for chemiluminescence

  • Consider using fluorescent secondary antibodies with digital imaging

• Antigen retrieval on membranes:

  • Brief methanol treatment of membranes

  • Incubation in 0.2% glutaraldehyde for 15 minutes before blocking

Each parameter should be systematically optimized while maintaining appropriate positive controls.

How can researchers validate the specificity of CCD7-1 antibody in their experimental system?

To rigorously validate CCD7-1 specificity in a new experimental system:

• Multiple detection methods:

  • Compare results across different applications (Western blot, IHC, IF)

  • Verify that the pattern of detection matches known biology of cadherin-7

• Molecular validation:

  • Perform immunoprecipitation followed by mass spectrometry to confirm target identity

  • Use RNA interference to knock down cadherin-7 and demonstrate reduced antibody signal

• Absorption controls:

  • Pre-incubate antibody with recombinant cadherin-7 before application

  • Show that this treatment abolishes specific staining

• Expression system validation:

  • Transfect cells with cadherin-7 expression constructs

  • Demonstrate increased CCD7-1 binding in transfected versus non-transfected cells

• Cross-reactivity assessment:

  • Test against cells expressing other cadherin family members

  • Demonstrate specificity for cadherin-7 versus related proteins

These validation steps provide comprehensive evidence for antibody specificity that meets publication standards.

How does the methodology for using CCD7-1 differ from approaches used with anti-CD7 CAR-T cell research?

The methodological approaches differ significantly based on the distinct biology and research applications:

While both involve antibodies, the research questions and methodological concerns are entirely different, with cadherin-7 research focusing on developmental biology and CD7 research on immunotherapy applications .

What are the advantages and limitations of CCD7-1 compared to molecular probes for cadherin-7?

When choosing between CCD7-1 antibody and molecular probes for cadherin-7 research:

The optimal approach often involves combining methodologies, using CCD7-1 for endogenous protein detection validated by complementary techniques .