wnt10a Antibody

What is Wnt10a and what biological functions does it serve?

Wnt10a functions as a ligand for the frizzled family of seven transmembrane receptors and operates primarily through the canonical Wnt/β-catenin signaling pathway. This protein plays essential roles in normal ectoderm development and is required for several developmental and maintenance processes including tooth development, postnatal development and maintenance of tongue papillae and sweat ducts, and normal hair follicle function. It regulates the proliferation of basal cells in tongue filiform papillae, plantar epithelium, and sweat ducts. Additionally, Wnt10a is required for normal expression of keratins in tongue papillae and KRT9 in foot plant epithelium .

From a molecular standpoint, Wnt10a influences cellular behavior by modulating the Wnt/β-catenin pathway, which regulates gene expression through nuclear translocation of β-catenin. This process ultimately affects downstream targets including cyclin D1 and c-myc, which control cell proliferation, differentiation, and survival .

What types of Wnt10a antibodies are available for research applications?

Commercially available Wnt10a antibodies include:

• Rabbit polyclonal antibodies: These are the most common type, generated by immunizing rabbits with synthetic peptides corresponding to regions within the human Wnt10a protein. Examples include ab106522 and ab189015, which are suitable for various applications including Western blot (WB), immunohistochemistry on paraffin-embedded tissues (IHC-P), and immunocytochemistry/immunofluorescence (ICC/IF) .

• Species reactivity: Available antibodies show reactivity primarily with human and mouse Wnt10a, though some may cross-react with rat samples due to sequence homology. When selecting an antibody, researchers should verify species reactivity based on their experimental model .

The choice of antibody depends on the specific application, species of interest, and epitope recognition requirements. Validation data, including Western blots showing the expected band size of approximately 46 kDa and positive control tissues (such as skeletal muscle), should be consulted when selecting an antibody for a particular research application .

How should researchers validate Wnt10a antibodies for their specific applications?

A systematic approach to Wnt10a antibody validation should include:

• Western blot analysis: Confirm the antibody detects a band at the expected molecular weight (approximately 46 kDa for Wnt10a). Testing at different antibody concentrations (e.g., 1 μg/mL and 2 μg/mL) helps determine optimal dilution ratios .

• Positive and negative control tissues: Include tissues known to express Wnt10a (e.g., skeletal muscle) as positive controls. For negative controls, use tissues with low/no Wnt10a expression or perform antibody ablation using competitive peptides (e.g., sc-69135P can abolish sc-69135 staining) .

• Immunohistochemical characteristics: Verify expected cellular localization patterns. Wnt10a typically shows cytoplasmic expression in target cells, with higher expression in certain tissues like RCC compared to normal renal tubular cells .

• RNA interference: Transfection with Wnt10a siRNA should reduce antibody signal in Western blot and immunostaining, confirming specificity .

• Overexpression systems: Transfection with Wnt10a expression vectors should increase antibody signal proportionally .

These validation approaches ensure that observed staining patterns represent genuine Wnt10a detection rather than non-specific binding or artifacts.

What are the optimal protocols for using Wnt10a antibodies in immunohistochemistry?

For successful immunohistochemical detection of Wnt10a, researchers should follow these methodological guidelines:

Paraffin-embedded tissue sections:

• Section thickness: 4 μm sections are recommended

• Blocking: Use 5-10% goat serum (for rabbit primary antibodies) for 30-60 minutes at room temperature

• Antigen retrieval: Heat-mediated antigen retrieval in citrate buffer (pH 6.0) is essential

• Primary antibody concentration: 5-10 μg/ml, with incubation for 1-2 hours at room temperature

• Secondary detection: Polymer HRP Detection System/DAB is effective for visualization

• Counterstaining: Hematoxylin provides good nuclear contrast

Immunofluorescence protocol:

• Primary antibody concentration: 10-20 μg/ml for cultured cells

• Secondary antibody: AlexaFluor®555-conjugated goat anti-rabbit IgG (1:400 dilution)

• Incubation time: 1 hour at room temperature for both primary and secondary antibodies

For confirmation of specificity, researchers should consider performing antibody ablation experiments using competitive peptides. In published studies, antibody ablation with WNT10A peptide (sc-69135P) successfully abolished the staining pattern of sc-69135, confirming specificity .

How can Wnt10a antibodies be effectively used in Western blot analysis?

For optimal Western blot detection of Wnt10a:

• Protein extraction: Standard lysis buffers containing protease inhibitors are suitable for extracting Wnt10a from tissues and cell lines

• Protein loading: 15-20 μg of total protein per lane is typically sufficient

• Antibody concentration: 1-2 μg/mL provides good signal-to-noise ratio

• Expected band size: 46 kDa is the predicted molecular weight for Wnt10a

• Positive controls: RAW264.7 cell lysates have been validated as positive controls

Protocol optimization considerations:

• Different antibody concentrations (e.g., 1 μg/mL vs. 2 μg/mL) should be tested to determine optimal signal

• Blocking with 5% non-fat dry milk or BSA in TBST is typically effective

• ECL-based detection systems provide adequate sensitivity for most applications

When interpreting Western blot results, researchers should be aware that post-translational modifications may cause slight variations in the observed molecular weight. Additionally, validation through siRNA knockdown of Wnt10a can provide compelling evidence of antibody specificity .

What cell and tissue models are most appropriate for studying Wnt10a function?

Based on the research literature, the following models are suitable for investigating Wnt10a:

Cell lines:

• Renal cell carcinoma (RCC) lines:

  • 786-O and A498: Low endogenous Wnt10a expression, suitable for overexpression studies

  • RCC-1 and Caki-1: High endogenous Wnt10a expression, appropriate for knockdown experiments

• Normal cell lines:

  • HK-2 (human kidney cells): Low endogenous Wnt10a expression

• RAW264.7 macrophage cell line: Positive control for Wnt10a expression in Western blot analysis

Tissue models:

• Renal tissues: Clear cell RCC (CCRCC) tissues show high Wnt10a expression compared to adjacent normal tissues

• Skeletal muscle: Human and mouse skeletal muscle tissues show detectable Wnt10a expression

• Developmental models: Studies of tooth development, sweat ducts, and hair follicles

Experimental approaches:

• Gain-of-function: Transfection with pcDNA-WNT10A vector allows overexpression studies

• Loss-of-function: WNT10A siRNA transfection (recommended concentration: 10 nM) provides effective knockdown

• Reporter assays: TCF/LEF luciferase reporters can measure canonical Wnt pathway activation downstream of Wnt10a

These models have proven valuable for investigating various aspects of Wnt10a biology, including its roles in development, tissue maintenance, and cancer progression .

How does Wnt10a contribute to oncogenic processes, and what experimental designs best elucidate these mechanisms?

Wnt10a has been implicated in oncogenic processes, particularly in renal cell carcinoma (RCC). Research has revealed several key mechanisms:

Oncogenic mechanisms of Wnt10a:

• Activation of the canonical Wnt/β-catenin pathway

• Upregulation of downstream targets including cyclin D1 and c-myc

• Enhancement of cancer cell migration, invasion, and colony formation

Experimental designs to investigate Wnt10a's oncogenic role:

• Expression analysis in patient samples:

  • Immunohistochemical analysis of tumor tissue microarrays comparing Wnt10a expression in tumor vs. normal tissue

  • Correlation of Wnt10a expression with clinicopathological parameters and patient survival

  • Co-expression analysis with β-catenin, cyclin D1, and c-myc to establish pathway activation

• Functional assays:

  • Migration assays: Wound-healing and transwell assays to assess cell motility

  • Invasion assays: Matrigel-coated transwell assays (recommended coating: 1 mg/mL Matrigel)

  • Colony formation assays: Soft agar colony formation to evaluate anchorage-independent growth

  • Cell proliferation assays: MTT or BrdU incorporation

• Molecular pathway analysis:

  • TCF/LEF reporter assays to measure canonical Wnt pathway activation

  • Western blot and immunocytochemistry to assess nuclear translocation of β-catenin

  • qRT-PCR for downstream target gene expression

• Rescue experiments:

  • Simultaneous knockdown of β-catenin in Wnt10a-overexpressing cells to determine pathway dependence

  • Combined analysis of survival data based on expression of Wnt10a, nuclear β-catenin, and cyclin D1

Research has demonstrated that patients with higher expression of Wnt10a, nuclear β-catenin, and nuclear cyclin D1 have significantly poorer prognosis in RCC, with a cumulative dose effect. This suggests that these markers together provide stronger prognostic value than any single marker alone .

What is the relationship between Wnt10a and transcriptional regulation in developmental processes?

Wnt10a plays critical roles in developmental processes through complex transcriptional regulatory networks:

Developmental roles of Wnt10a:

• Ectoderm development

• Tooth development

• Tongue papillae and sweat duct development and maintenance

• Hair follicle function

Transcriptional regulation of Wnt10a:
Research has shown that BATF (Basic Leucine Zipper ATF-Like Transcription Factor) regulates Wnt10a expression during B cell stimulation. BATF expression leads to dramatic reduction of Wnt10a mRNA, suggesting a repressive role. Interestingly, this regulation appears to be time-dependent, with BATF expression at 6 hours being sufficient to repress Wnt10a, even without sustained BATF expression afterwards .

Experimental approaches to study transcriptional regulation:

• Analysis of temporal expression patterns following stimulation

• Rescue experiments using viral vectors (e.g., MSCV-Thy1.1) expressing Wnt10a

• Inducible expression systems to control timing of transcription factor activity

• ChIP-seq to identify direct binding of transcription factors to Wnt10a regulatory regions

Understanding the transcriptional regulation of Wnt10a provides insights into how its expression is precisely controlled during development and in response to various stimuli, which is crucial for normal tissue development and homeostasis .

How does Wnt10a interact with the Wnt/β-catenin signaling pathway at the molecular level?

Wnt10a functions as a ligand in the canonical Wnt/β-catenin signaling pathway, with several important molecular interactions:

Molecular interactions and pathway components:

• Receptor binding: Wnt10a binds to members of the frizzled family of seven transmembrane receptors

• β-catenin stabilization: Upon Wnt10a binding, the destruction complex is inhibited, leading to cytoplasmic accumulation of β-catenin

• Nuclear translocation: Accumulated β-catenin translocates to the nucleus

• Transcriptional activation: Nuclear β-catenin interacts with TCF/LEF transcription factors to activate target genes

• Downstream targets: Key targets include cyclin D1 and c-myc, which regulate cell proliferation and other cellular processes

Experimental evidence for pathway activation:

• Immunohistochemical analysis shows that tissues with high Wnt10a expression also exhibit increased cytoplasmic and nuclear β-catenin accumulation

• Forced Wnt10a expression in cell lines increases nuclear β-catenin, cyclin D1, and c-myc levels

• TCF/LEF reporter assays demonstrate that Wnt10a overexpression significantly induces luciferase activity

• siRNA knockdown of β-catenin reduces the effects of Wnt10a overexpression on cyclin D1 and c-myc expression

• Functional assays show that β-catenin knockdown attenuates the effects of Wnt10a on cell migration, invasion, and colony formation

These findings confirm that Wnt10a functions primarily through the canonical Wnt/β-catenin pathway to regulate gene expression and cellular processes. The simultaneous analysis of Wnt10a, β-catenin, and downstream targets provides a more comprehensive understanding of pathway activity than any single marker alone .

What are common technical challenges when working with Wnt10a antibodies and how can they be addressed?

Researchers frequently encounter several challenges when working with Wnt10a antibodies:

Challenge 1: Non-specific staining

• Potential causes: Insufficient blocking, excessive antibody concentration, cross-reactivity

• Solutions:

  • Optimize blocking conditions (10% goat serum for 1 hour at room temperature)

  • Titrate antibody concentration (test 1-10 μg/ml range)

  • Include peptide competition controls to confirm specificity

  • Use knockout or knockdown samples as negative controls

Challenge 2: Weak or absent signal

• Potential causes: Inadequate antigen retrieval, suboptimal fixation, low target expression

• Solutions:

  • Optimize antigen retrieval (heat-mediated in citrate buffer, pH 6.0)

  • Test multiple antibodies targeting different epitopes of Wnt10a

  • Use positive control tissues (skeletal muscle has been validated)

  • Increase antibody incubation time (up to overnight at 4°C)

Challenge 3: Inconsistent Western blot results

• Potential causes: Protein degradation, inefficient transfer, suboptimal detection

• Solutions:

  • Use fresh samples with protease inhibitors

  • Optimize protein loading (15-20 μg recommended)

  • Test multiple antibody concentrations (1-2 μg/ml range)

  • Verify expected band size (46 kDa)

Challenge 4: Variability between experiments

• Potential causes: Inconsistent protocols, antibody batch variation

• Solutions:

  • Standardize protocols with detailed SOPs

  • Use the same antibody lot when possible

  • Include consistent positive and negative controls

  • Quantify results using image analysis software

For reproducible results, researchers should validate antibodies using multiple techniques and implement rigorous controls in each experiment .

What control samples are essential for validating Wnt10a antibody experiments?

A comprehensive control strategy is crucial for validating Wnt10a antibody experiments:

Positive controls:

• Tissue controls: Human and mouse skeletal muscle tissues have been validated for Wnt10a expression

• Cell line controls: RAW264.7 cell lysates for Western blot; RCC-1 and Caki-1 cells show high endogenous Wnt10a expression

• Overexpression controls: Cells transfected with pcDNA-WNT10A vector

Negative controls:

• Antibody omission: Primary antibody replaced with diluent only

• Peptide competition: Pre-incubation of antibody with blocking peptide (e.g., sc-69135P for sc-69135)

• Knockdown controls: Cells transfected with Wnt10a siRNA

• Low-expression tissues: Normal renal tubular cells show minimal Wnt10a expression

Pathway validation controls:

• β-catenin co-staining: To confirm pathway activation

• Downstream marker analysis: Cyclin D1 and c-myc expression

• Functional rescue: β-catenin siRNA co-transfection to revert Wnt10a-induced phenotypes

Technical controls:

• Multiple antibody concentrations: Testing 1 μg/ml and 2 μg/ml for Western blot

• Cross-validation: Using multiple antibodies targeting different epitopes

• Isotype controls: Non-specific antibodies of the same isotype and concentration

Incorporating these controls allows researchers to confidently interpret Wnt10a staining patterns and functional effects .

How can quantitative analysis of Wnt10a expression be standardized across studies?

Standardizing quantitative analysis of Wnt10a expression is essential for comparing results across studies:

Immunohistochemistry quantification:

• Histoscore method:

  • Calculate: Histoscore = positive cell percentage × intensity

  • Intensity scale: negative (0), weak (1), moderate (2), and strong (3)

  • Separate scoring for membranous, cytoplasmic, and nuclear staining

  • Score range: 0-300

• Digital image analysis:

  • Use calibrated software to measure staining intensity

  • Establish standardized thresholds for positive staining

  • Report both percentage of positive cells and mean intensity

Western blot quantification:

• Normalize Wnt10a band intensity to loading controls (β-actin, GAPDH)

• Use digital densitometry with linear range validation

• Report relative expression compared to control samples

RT-qPCR standardization:

• Use validated reference genes (GAPDH, β-actin, or multiple reference genes)

• Apply the ΔΔCt method for relative quantification

• Include efficiency controls and no-template controls

Reporting standards:

• Clearly document antibody details (source, catalog number, dilution)

• Describe image acquisition parameters (exposure time, gain)

• Detail quantification methodology

• Include representative images of all scoring categories

Implementing these standardized approaches facilitates meta-analysis and reproducibility across different studies investigating Wnt10a expression and function .

How does Wnt10a expression correlate with clinical outcomes in cancer patients?

Research on Wnt10a expression in cancer, particularly renal cell carcinoma (RCC), reveals significant correlations with clinical outcomes:

Expression patterns in cancer:

• RCC tissues show dramatically higher cytoplasmic Wnt10a expression compared to non-tumoral renal tissues

• Normal renal tubular cells from both cortex and medulla exhibit very low cytoplasmic Wnt10a expression

Correlation with pathway activation:
In RCC tissues with high Wnt10a expression:

• β-catenin shows higher intracellular accumulation (cytoplasmic and nuclear)

• c-myc and cyclin D1 show higher nuclear expression

• These patterns contrast with normal kidney tissue, which shows predominantly membranous β-catenin expression

Prognostic significance:
Kaplan-Meier analysis demonstrates an accumulated dose effect:

This accumulated effect supports the biological relevance of the Wnt10a/β-catenin/cyclin D1 axis in cancer progression and provides a rationale for using multiple markers in prognostic assessment .

What are the recommended experimental designs for studying Wnt10a function in developmental biology?

For investigating Wnt10a's role in developmental processes, researchers should consider these experimental approaches:

In vitro models:

• Organoid cultures: Tooth bud, hair follicle, or sweat gland organoids allow 3D study of developmental processes

• Primary cell cultures: Isolated from relevant tissues during developmental stages

• Differentiation assays: Assess the impact of Wnt10a manipulation on cellular differentiation markers

In vivo models:

• Conditional knockout models: Tissue-specific and temporally controlled deletion of Wnt10a

• Reporter mouse lines: Fluorescent or LacZ reporters driven by Wnt10a promoter to track expression patterns

• Lineage tracing: To follow the fate of Wnt10a-expressing cells during development

Molecular techniques:

• Single-cell RNA sequencing: To identify cell populations expressing Wnt10a and responding to Wnt signals

• ChIP-seq analysis: To identify transcription factors regulating Wnt10a expression

• CRISPR/Cas9 genome editing: For precise modification of Wnt10a or pathway components

Functional readouts:

• Morphological analysis: Detailed assessment of developmental phenotypes

• Immunostaining for differentiation markers: Keratin expression in epithelial structures

• Cell proliferation assays: EdU or BrdU incorporation to assess proliferative effects

• Pathway analysis: TCF/LEF reporter assays to measure canonical Wnt activity

Given Wnt10a's roles in ectoderm development, tooth development, tongue papillae maintenance, and hair follicle function, these experimental approaches allow comprehensive investigation of its developmental functions .

How can researchers effectively design Wnt10a gain-of-function and loss-of-function experiments?

Design of robust gain-of-function and loss-of-function experiments for Wnt10a requires careful consideration of several factors:

Gain-of-function approaches:

• Plasmid-based overexpression:

  • Vector: pcDNA-WNT10A has been validated for effective expression

  • Transfection: 3 μg of plasmid DNA typically achieves significant overexpression

  • Selection: G418 (200 μg/mL) can maintain expression in long-term experiments

  • Controls: Empty vector (pcDNA3.1) transfection is essential

• Viral delivery systems:

  • MSCV-based retroviruses provide effective delivery to dividing cells

  • Lentiviral systems offer broader tropism and can transduce non-dividing cells

  • Inducible expression systems allow temporal control of Wnt10a expression

Loss-of-function approaches:

• siRNA knockdown:

  • Concentration: 10 nM WNT10A siRNA has been validated for effective knockdown

  • Duration: 72 hours post-transfection for optimal protein reduction

  • Controls: Scrambled siRNA controls are essential

  • Validation: Western blot and RT-qPCR to confirm knockdown efficiency

• CRISPR/Cas9 knockout:

  • Complete gene knockout for long-term studies

  • Multiple guide RNAs targeting different exons

  • Single-cell cloning to obtain homogeneous knockout populations

Experimental validation:

• Proof of manipulation:

  • Western blot showing Wnt10a protein levels

  • RT-qPCR for mRNA expression

  • Immunocytochemistry to confirm cellular expression patterns

• Pathway activation confirmation:

  • β-catenin nuclear localization by immunostaining or nuclear/cytoplasmic fractionation

  • TCF/LEF reporter assays to measure downstream pathway activation

  • Expression of known target genes (cyclin D1, c-myc) by Western blot and RT-qPCR

• Functional readouts:

  • Cell migration assays (wound healing, transwell migration)

  • Invasion assays (Matrigel-coated transwells)

  • Colony formation assays (soft agar)

  • Proliferation assays (MTT, BrdU incorporation)

• Rescue experiments:

  • Co-transfection of WNT10A with β-catenin siRNA to test pathway dependence

  • Re-expression of Wnt10a in knockout models to confirm specificity

These experimental designs provide comprehensive approaches to investigate Wnt10a function in various biological contexts, from development to cancer progression .