CDH5 Monoclonal Antibody

What is CDH5 and why is it important in biomedical research?

CDH5 (Cadherin-5) is an endothelial-specific adhesion molecule that constitutes a major component of endothelial junctions. It plays critical roles in maintaining vascular integrity, regulating endothelial permeability, and mediating endothelial cell adhesion. In research contexts, CDH5 serves as a key marker for endothelial cells and has significant implications in vascular biology, angiogenesis research, and cancer metastasis studies. The protein has a calculated molecular weight of approximately 87-88 kDa and contains extracellular calcium-binding domains typical of cadherins . Studies have shown that CDH5 expression in tumor cells is associated with hematogenous recurrence and shorter progression-free intervals in certain cancers, highlighting its importance as a potential biomarker and therapeutic target .

How should I select the appropriate CDH5 monoclonal antibody for my research?

When selecting a CDH5 monoclonal antibody, consider the following methodological approach:

• Target species reactivity: Determine whether you need an antibody that recognizes human, mouse, or other species-specific CDH5. Available antibodies include mouse anti-human CDH5 and rabbit anti-mouse CDH5 variants .

• Epitope specificity: Some antibodies recognize calcium-independent extracellular epitopes (e.g., clone 55-7H1), which may be advantageous for certain applications .

• Validated applications: Ensure the antibody has been validated for your specific application:

  • For Western blotting: Both mouse and rabbit monoclonals are available with validated protocols

  • For immunohistochemistry: Select antibodies specifically validated for IHC-F or ICC

  • For flow cytometry: Choose antibodies with demonstrated specificity in flow applications

  • For immunoprecipitation: Verify IP validation data is available

• Clone specificity: Different clones (such as 55-7H1, OTI1F4, IIC-3) may have different performance characteristics depending on application and epitope recognition .

What is the difference between CDH5 and other cadherins in experimental contexts?

CDH5 differs from other cadherins in several significant ways that impact experimental design:

• Tissue specificity: Unlike CDH1 (E-cadherin) which is broadly expressed in epithelial tissues, CDH5 expression is strictly endothelial-specific, making it a highly selective marker for endothelial cells and vascular structures .

• Junction dynamics: CDH5 undergoes phosphorylation under vascular permeability-increasing conditions, promoting rapid and reversible internalization. This dynamic regulation differs from other cadherins and is crucial for researchers studying vascular permeability .

• Cancer implications: While many cadherins function primarily in cell adhesion, CDH5 has distinct roles in cancer progression. Studies show CDH5 is significantly associated with hematogenous recurrence in advanced gastric cancer, suggesting unique functions in tumor cell dissemination through blood vessels .

• Functional domains: When designing domain-specific studies, it's important to note that CDH5's extracellular domain structure has specific binding properties that differ from other cadherins, particularly in calcium-dependent versus calcium-independent interactions .

What are the optimal conditions for using CDH5 antibodies in Western blotting?

For optimal Western blotting with CDH5 monoclonal antibodies, follow these methodological guidelines:

• Sample preparation:

  • For endothelial cells or tissues with known CDH5 expression (such as lung tissue for mouse studies), prepare lysates under reducing conditions

  • Load approximately 30 μg of protein per lane for optimal detection

• Gel electrophoresis parameters:

  • Use 5-20% gradient SDS-PAGE gels for optimal separation

  • Run at 70V for stacking gel and 90V for resolving gel

  • Expected molecular weight visualization: approximately 88 kDa

• Transfer and blocking:

  • Transfer to nitrocellulose membrane at 150 mA for 50-90 minutes

  • Block with 5% non-fat milk in TBS for 1.5 hours at room temperature

• Antibody incubation:

  • For rabbit anti-mouse CDH5: Dilute 1:500-1:1000 in blocking buffer and incubate overnight at 4°C

  • For mouse anti-human CDH5: Similar dilutions are recommended

  • Wash with TBS-0.1% Tween, 3 times for 5 minutes each

• Detection system:

  • Use appropriate HRP-conjugated secondary antibody (e.g., goat anti-rabbit IgG-HRP) at 1:500 dilution

  • Develop using enhanced chemiluminescence detection systems

Positive controls should include known CDH5-expressing samples such as HUVEC cells for human studies or mouse lung tissue lysates for mouse studies .

How can I optimize CDH5 antibody use in flow cytometry for endothelial cell research?

For flow cytometric analysis of CDH5 expression, implement these methodological approaches:

• Cell preparation:

  • Use single-cell suspensions of endothelial cells (such as HUVECs for human studies)

  • Ensure viability >90% for optimal results

  • For adherent endothelial cells, use gentle enzymatic dissociation methods that preserve surface epitopes

• Antibody concentration optimization:

  • Begin with the recommended concentration of 1-4 μg/ml for anti-CD144 antibodies

  • Perform titration experiments (0.5, 1, 2, 4, 8 μg/ml) to determine optimal signal-to-noise ratio for your specific cell type

• Staining protocol:

  • For surface CDH5: Perform staining on ice in buffer containing 1% BSA and 0.1% sodium azide

  • Include appropriate isotype control (Mouse IgG1 kappa for clone 55-7H1)

  • Use secondary detection with fluorophore-conjugated anti-mouse antibodies such as GAM APC

• Gating strategy:

  • Gate first on viable single cells

  • Compare stained population with isotype control and unstained cells

  • CDH5 typically shows surface localization with distinct positive population

• Controls essential for validation:

  • Positive control: Confirmed CDH5-positive endothelial cells

  • Negative control: Non-endothelial cells

  • FMO (Fluorescence Minus One) controls for multicolor panels

The flow cytometry data should show clear separation between CDH5-positive endothelial cells and controls, with minimal background staining, as demonstrated in validation studies with HUVEC cells .

What methodological considerations are important for immunohistochemistry using CDH5 antibodies?

When performing immunohistochemistry with CDH5 antibodies, consider these methodological aspects:

• Tissue preparation:

  • For frozen sections (IHC-F): Snap freeze tissues in OCT compound and prepare 5-8 μm sections

  • Fix sections in cold acetone for 10 minutes to preserve CDH5 epitopes

  • For certain antibodies validated for IHC, formalin-fixed paraffin-embedded tissues may be used with appropriate antigen retrieval

• Antigen retrieval optimization:

  • For FFPE sections: Test both citrate buffer (pH 6.0) and EDTA buffer (pH 9.0)

  • Heat-induced epitope retrieval methods are typically more effective than enzymatic methods for CDH5

  • Optimize retrieval time (10-20 minutes) based on tissue type and fixation duration

• Antibody dilution and incubation:

  • For OTI1F4 clone: Use at 1:150 dilution

  • Incubate overnight at 4°C in humid chamber for optimal staining

  • For mouse antibodies on mouse tissues, use mouse-on-mouse blocking kits to reduce background

• Detection systems:

  • For brightfield microscopy: Use polymer detection systems rather than ABC method for enhanced sensitivity

  • For fluorescence: Select secondary antibodies with minimal spectral overlap with other channels

  • Include DAPI counterstain to visualize nuclei in fluorescent applications

• Controls and interpretation:

  • Positive control: Include known CDH5-positive tissues (blood vessels in any vascularized tissue)

  • Negative control: Omit primary antibody or use isotype control

  • Expected pattern: Distinct membrane staining at endothelial cell junctions

  • Quantification approach: Measure vessel density or junction integrity using image analysis software

This approach ensures specific detection of CDH5 in vascular structures while minimizing background and non-specific staining.

How can CDH5 antibodies be used to study endothelial-to-mesenchymal transition (EndMT)?

EndMT is a critical process in development and disease where endothelial cells transition to a mesenchymal phenotype. CDH5 antibodies serve as valuable tools for investigating this process:

• Experimental design for EndMT studies:

  • Establish baseline CDH5 expression in normal endothelial cells via Western blot and immunofluorescence

  • Induce EndMT using appropriate stimuli (TGF-β, inflammatory cytokines, hypoxia)

  • Monitor temporal changes in CDH5 expression and localization

• Multi-marker analysis approach:

  • Combine CDH5 antibodies with mesenchymal markers (α-SMA, FSP1, N-cadherin)

  • Use flow cytometry to quantify population shifts during transition

  • Implement co-immunoprecipitation with CDH5 antibodies to track changing protein interactions during EndMT

• Functional assays:

  • Correlate CDH5 downregulation with increased permeability using transwell assays

  • Use CDH5 antibodies to block function and assess impact on EndMT progression

  • Implement proximity ligation assays to study interactions between CDH5 and regulatory proteins

• Validation in disease models:

  • Several studies have implicated Lysyl oxidase-like 2 as a regulator of angiogenesis through modulation of EndMT, where CDH5 serves as a critical marker for monitoring this transition

  • Monitor CDH5 expression patterns in fibrotic diseases where EndMT contributes to pathogenesis

This methodological framework allows researchers to comprehensively analyze the dynamics of EndMT using CDH5 as a key endothelial marker that is typically lost during transition to mesenchymal phenotype.

What methodological approaches can be used to study the role of CDH5 in cancer metastasis?

Based on research showing CDH5 as a significant risk factor for hematogenous metastasis, these methodological approaches can be implemented:

• Expression analysis in clinical samples:

  • Use validated CDH5 antibodies for IHC staining of primary tumors and metastatic lesions

  • Develop standardized scoring systems based on staining intensity and localization patterns

  • Correlate expression levels with clinical outcomes including metastasis-free survival

• Functional metastasis assays:

  • Establish cell lines with differential CDH5 expression (overexpression, knockdown)

  • Validate expression changes via Western blot using monoclonal antibodies

  • Perform transendothelial migration assays to assess how CDH5 expression affects tumor cell transit through endothelial barriers

  • Implement in vivo metastasis models with bioluminescence imaging to track CDH5-expressing cells

• Mechanistic studies:

  • Use CDH5 antibodies for chromatin immunoprecipitation to identify transcriptional regulation

  • Perform co-immunoprecipitation to identify CDH5-interacting partners in tumor cells

  • Analyze phosphorylation status of CDH5 in tumor cells using phospho-specific antibodies

• Translational relevance:

  • Develop protocols for using CDH5 antibodies as prognostic tools in cancer patients

  • Standardize CDH5 detection methods for potential clinical implementation

  • Research from gastric cancer studies demonstrates that high CDH5 expression was associated with significantly shorter progression-free intervals (hazard ratio 2.2) and independently predicted hematogenous recurrence (odds ratio 3.9)

This comprehensive approach enables researchers to investigate the multifaceted roles of CDH5 in cancer progression and metastasis.

How can phosphorylation status of CDH5 be assessed in experimental models?

The phosphorylation status of CDH5 is critical for its internalization and regulation of vascular permeability. Use these methodological approaches to assess phosphorylation:

• Western blot analysis:

  • Use phospho-specific antibodies targeting key CDH5 phosphorylation sites

  • Implement phosphatase inhibitors in lysis buffers to preserve phosphorylation status

  • Run parallel blots with total CDH5 antibodies to calculate phospho/total ratios

  • Use lambda phosphatase treatment as a negative control to confirm phospho-specificity

• Immunoprecipitation strategy:

  • Use validated CDH5 monoclonal antibodies for immunoprecipitation (IP dilution 1:50)

  • Perform IP under non-denaturing conditions to maintain phosphoepitopes

  • Probe with anti-phosphotyrosine or phospho-serine/threonine antibodies

  • Alternatively, immunoprecipitate with phospho-specific antibodies and probe with total CDH5

• Immunofluorescence approaches:

  • Use phospho-specific CDH5 antibodies for immunofluorescence (IF dilution 1:500-1:1000)

  • Implement confocal microscopy to assess subcellular localization of phosphorylated CDH5

  • Counterstain with total CDH5 to determine proportion of phosphorylated protein

  • Use physiological stimuli known to induce CDH5 phosphorylation (VEGF, histamine, thrombin)

• Functional correlation:

  • Correlate phosphorylation status with endothelial barrier function using TEER measurements

  • Implement phosphomimetic and phospho-dead CDH5 mutants to validate antibody specificity

  • Research indicates that CDH5 phosphorylation is particularly prevalent in capillaries and veins and occurs under conditions that increase vascular permeability

This multifaceted approach enables comprehensive assessment of CDH5 phosphorylation in relation to its functional status in regulating vascular integrity.

What are common technical issues when using CDH5 antibodies and how can they be resolved?

When working with CDH5 monoclonal antibodies, researchers may encounter several technical challenges. Here are methodological solutions for common issues:

• Low signal in Western blotting:

  • Increase protein loading to 30-50 μg per lane

  • Optimize primary antibody concentration (try 1:250 instead of 1:500)

  • Extend primary antibody incubation to overnight at 4°C

  • Use enhanced chemiluminescent detection systems with longer exposure times

  • Ensure sample preparation preserves CDH5 integrity (add protease inhibitors)

• Non-specific binding in immunohistochemistry:

  • Implement additional blocking steps (10% serum from secondary antibody species)

  • Reduce antibody concentration and extend incubation time

  • Perform antigen retrieval optimization experiments

  • For mouse tissues, use mouse-on-mouse blocking kits when using mouse primary antibodies

  • Include validated positive and negative controls in each experiment

• Poor flow cytometry resolution:

  • Optimize antibody concentration through titration experiments

  • Ensure cells remain viable throughout processing (>90% viability)

  • Use gentler cell dissociation methods to preserve surface epitopes

  • Implement proper compensation when using multiple fluorophores

  • Pre-clear cell suspensions to remove aggregates

• Cross-reactivity concerns:

  • Validate antibody specificity using knockout/knockdown controls

  • Note that some clones (e.g., 55-7H1) have been tested for non-cross-reactivity with specific antigens like Thy-1.1

  • When working with mixed cell populations, include appropriate lineage markers

• Antibody stability issues:

  • Adhere to proper storage conditions (store at -20°C long-term; 4°C for frequent use)

  • Avoid repeated freeze-thaw cycles which can degrade antibody quality

  • Aliquot antibodies upon receipt to minimize freeze-thaw cycles

  • Add carrier protein (BSA) if diluting antibodies for longer storage

Implementing these technical approaches will enhance the reliability and reproducibility of experiments using CDH5 monoclonal antibodies.

How should I validate the specificity of a CDH5 monoclonal antibody for my experimental system?

A comprehensive validation strategy for CDH5 monoclonal antibodies should include:

• Positive and negative control tissues/cells:

  • Positive controls: Endothelial cells (HUVECs for human studies), vascular tissues (lung tissue for mouse studies)

  • Negative controls: Cell lines known not to express CDH5 (fibroblasts, epithelial cells)

  • Perform parallel experiments with multiple antibody clones to verify consistent staining patterns

• Molecular validation approaches:

  • siRNA/shRNA knockdown of CDH5 followed by antibody testing

  • CRISPR-Cas9 knockout models as definitive negative controls

  • Transfection of CDH5-negative cells with CDH5 expression constructs

  • Peptide blocking experiments using the immunizing peptide when available

• Technical validation methods:

  • Multiple application testing (if antibody is validated for multiple applications)

  • Antibody titration to determine optimal signal-to-noise ratio

  • Cross-species reactivity assessment if working with models from different species

  • Testing under native and denatured conditions to confirm epitope accessibility

• Literature cross-validation:

  • Compare results with published literature using the same or different clones

  • Reference established staining patterns and molecular weights

  • Note that validated antibodies should detect CDH5 at approximately 87-88 kDa

• Application-specific validation:

  • For Western blot: Confirm single band at expected molecular weight

  • For IHC/ICC: Verify membrane localization at cell-cell junctions

  • For flow cytometry: Demonstrate population shift compared to isotype control

  • For IP: Confirm enrichment of target protein in immunoprecipitate versus input

Thorough validation ensures experimental reliability and facilitates accurate interpretation of results across diverse experimental systems.

How can CDH5 monoclonal antibodies be used to investigate vascular permeability in disease models?

CDH5 serves as a critical regulator of vascular permeability, making CDH5 antibodies valuable tools for investigating barrier dysfunction in disease:

• Quantitative assessment methods:

  • Use immunofluorescence with CDH5 antibodies to visualize adherens junction integrity

  • Implement live-cell imaging with non-blocking fluorescently tagged CDH5 antibodies

  • Quantify gap formation and junction discontinuity using image analysis software

  • Track CDH5 internalization under permeability-inducing conditions

• Correlation with functional parameters:

  • Pair CDH5 staining with permeability assays (FITC-dextran, Evans blue)

  • Measure transendothelial electrical resistance (TEER) concurrent with CDH5 localization

  • Track CDH5 phosphorylation status which promotes its internalization during increased permeability

  • Correlate with in vivo vascular leakage in relevant disease models

• Mechanistic investigations:

  • Use CDH5 antibodies in proximity ligation assays to study interactions with regulatory proteins

  • Implement FRAP (fluorescence recovery after photobleaching) to study CDH5 dynamics

  • Assess CDH5 clustering and organization at cell junctions during barrier disruption

  • Research indicates that p120 catenin binding stabilizes CDH5 at the membrane, reducing vascular permeability

• Disease-specific applications:

  • Inflammatory conditions: Assess CDH5 redistribution during acute inflammation

  • Tumor vasculature: Examine heterogeneity of CDH5 distribution in cancer vessels

  • Stroke models: Investigate CDH5 disruption during blood-brain barrier breakdown

  • Pulmonary edema: Monitor CDH5 dynamics during lung injury

This methodological framework enables comprehensive analysis of how CDH5 regulates vascular permeability in both physiological and pathological contexts.

What approaches can be used to study CDH5's role in tumor angiogenesis?

CDH5 plays critical roles in angiogenesis, and these methodological approaches can elucidate its functions in tumor vascularization:

• Tumor vessel characterization:

  • Use CDH5 antibodies to assess vessel density and morphology in tumor sections

  • Implement dual staining with proliferation markers to identify actively growing vessels

  • Analyze vessel maturity by co-staining with pericyte markers (α-SMA, desmin)

  • Quantify abnormal junction patterns characteristic of tumor vessels

• 3D angiogenesis models:

  • Utilize CDH5 antibodies in 3D endothelial sprouting assays

  • Track CDH5 redistribution during tip/stalk cell specification

  • Assess impact of tumor-derived factors on CDH5 localization and integrity

  • Implement time-lapse imaging with non-blocking fluorescently tagged antibodies

• Mechanistic investigation approaches:

  • Study CDH5's interaction with VEGFR2 signaling using co-immunoprecipitation

  • Analyze CDH5 phosphorylation status in response to angiogenic factors

  • Assess CDH5 endocytosis and recycling during active angiogenesis

  • Research indicates that Lysyl oxidase-like 2 regulates angiogenesis through modulation of endothelial-to-mesenchymal transition, where CDH5 serves as a key marker

• Therapeutic targeting strategies:

  • Test effects of vascular normalizing agents on CDH5 junction organization

  • Evaluate CDH5-blocking antibodies as potential anti-angiogenic agents

  • Implement CDH5 antibodies as targeting vehicles for tumor vessel-specific delivery

  • Monitor CDH5 as a biomarker for treatment response in anti-angiogenic therapy

This multimodal approach enables comprehensive analysis of CDH5's contributions to tumor angiogenesis, with implications for both basic research and therapeutic development.

What are emerging applications for CDH5 monoclonal antibodies in research and clinical translation?

CDH5 monoclonal antibodies continue to evolve in their research applications and clinical potential:

• Advanced imaging applications:

  • Super-resolution microscopy to study nanoscale CDH5 organization at endothelial junctions

  • Intravital imaging with non-blocking fluorescently labeled antibodies to track junction dynamics in vivo

  • Correlative light-electron microscopy to link CDH5 distribution with ultrastructural features

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

• Biomarker development potential:

  • Soluble CDH5 detection in patient samples as a biomarker of vascular damage

  • Tissue-based CDH5 expression patterns as predictors of cancer metastasis risk

  • Research in gastric cancer indicates that high CDH5 expression is associated with hematogenous recurrence (odds ratio 3.9) and shorter progression-free intervals (hazard ratio 2.2)

  • Monitoring CDH5 dynamics during therapy to assess vascular normalization

• Therapeutic targeting approaches:

  • Development of function-blocking antibodies to modulate vascular permeability

  • Antibody-drug conjugates targeting CDH5 for vascular-specific therapy

  • CDH5-targeted nanoparticles for endothelial-specific drug delivery

  • Combination strategies targeting both CDH5 and VEGF pathways

• Single-cell applications:

  • Integration with single-cell proteomics and transcriptomics

  • Analysis of CDH5 heterogeneity in distinct vascular beds

  • Correlation of CDH5 expression with endothelial subtypes and functional states

  • Development of computational methods to quantify subtle changes in junction architecture

These emerging applications highlight the continued importance of well-validated CDH5 monoclonal antibodies in advancing both fundamental vascular biology and translational research in multiple disease contexts.