FZD9 Antibody

What is FZD9 and what biological systems express this receptor?

FZD9 (also known as CD349) belongs to the frizzled family of 7-transmembrane domain receptors that mediate Wnt signaling by pairing with low-density lipoprotein-like receptors 5 and 6. FZD9 is expressed predominantly in brain, testis, eye, skeletal muscle, and kidney tissues . The gene encoding FZD9 is located within chromosome 7q11.23, a region that is commonly deleted in Williams-Beuren syndrome (WBS) . When studying FZD9 expression patterns, researchers should consider tissue-specific variations in expression levels, as detection sensitivity may require optimization based on the target tissue.

For antibody-based detection of FZD9, researchers should note that expression levels vary significantly between tissues and developmental stages. When designing experiments targeting multiple tissues, consider using positive control samples from high-expression tissues like brain or testis to validate antibody performance before examining tissues with potentially lower expression levels.

How do I optimize FZD9 antibody-based detection in immunofluorescence applications?

When using FZD9 antibodies for immunofluorescence applications, several methodological considerations can improve detection specificity and sensitivity. Based on published protocols, the following approach is recommended:

• Fixation: For most cell types, 4% paraformaldehyde for 15-20 minutes at room temperature preserves FZD9 epitopes while maintaining cellular architecture.

• Permeabilization: Use 0.2-0.3% Triton X-100 for 10 minutes, as stronger detergents may disrupt the transmembrane structure of FZD9.

• Blocking: A 5-10% solution of normal serum (matching the secondary antibody host) with 1% BSA for 1-2 hours significantly reduces background.

• Primary antibody incubation: Optimal dilutions typically range from 1:100 to 1:500 depending on the specific antibody. Overnight incubation at 4°C generally yields better results than shorter incubations at room temperature .

• Controls: Always include both positive controls (tissues known to express FZD9, such as brain sections) and negative controls (FZD9-knockout tissues or isotype controls) to validate specificity.

When examining FZD9 in cell culture models of oncogenesis, researchers have successfully used this approach to track changes in receptor expression following MYC activation, revealing critical insights into Wnt signaling dynamics in cancer progression .

What are the methodological considerations for FZD9 antibody validation?

Proper validation of FZD9 antibodies is essential for reliable experimental outcomes. A comprehensive validation approach should include:

• Specificity testing: Verify the absence of signal in FZD9 knockout or knockdown models. The Fzd9-/- mouse model has been extensively characterized and serves as an excellent negative control for antibody validation .

• Cross-reactivity assessment: Test against other frizzled family members, particularly those with high sequence homology to FZD9. This is crucial as FZD9 shares structural similarities with other frizzled receptors, particularly in the cysteine-rich domain.

• Multiple detection methods: Confirm consistent detection across different techniques (Western blot, immunofluorescence, flow cytometry) to ensure epitope accessibility in various experimental conditions.

• Recombinant protein controls: Use purified recombinant FZD9 protein as a positive control and for antibody pre-absorption experiments to confirm binding specificity.

• Application-specific validation: When using FZD9 antibodies to isolate mesenchymal stem cells, validate the phenotype of antibody-positive cells by confirming co-expression of established MSC markers like CD9, CD63, and CD90 .

Researchers should document validation steps thoroughly and consider sensitivity limitations when interpreting negative results, particularly in tissues with naturally low FZD9 expression.

How can FZD9 antibodies be employed in studies of Myc-driven oncogenesis?

FZD9 has been identified as a direct Myc target gene through expression and ChIP analysis, making it a valuable marker in Myc-driven tumorigenesis research . When designing experiments to study this relationship, consider these methodological approaches:

• Temporal expression analysis: Use FZD9 antibodies to track receptor expression changes at different timepoints following Myc activation. In pancreatic insulinoma models, researchers observed significant upregulation of FZD9 within 3 days of Myc activation, preceding phenotypic changes in proliferation .

• Co-immunoprecipitation: Employ FZD9 antibodies to isolate protein complexes and identify binding partners that may connect Myc and Wnt signaling pathways. This approach has revealed critical insights into the molecular mechanisms through which FZD9 mediates Myc-dependent oncogenic transformation.

• Tissue microarray analysis: For translational studies, FZD9 antibodies can be applied to tumor tissue microarrays to correlate expression with clinical outcomes and Myc status.

• Functional blocking studies: Use neutralizing FZD9 antibodies to disrupt Wnt signaling in Myc-driven tumor models. Research has shown that blocking FZD9 can significantly impair sustained tumor expansion and affect Myc-related global gene expression patterns .

When interpreting results, note that FZD9's role in tumorigenesis appears context-dependent, as it has demonstrated both oncogenic properties in some cancers (gastric, osteosarcoma, astrocytoma) and tumor suppressor activity in others (acute myeloid leukemia, non-small cell lung cancer) .

What protocols are optimal for using FZD9 antibodies in flow cytometry for stem cell isolation?

FZD9 (CD349) has emerged as a valuable marker for the prospective isolation of mesenchymal stem cells. For optimal flow cytometry protocols when isolating FZD9+ cells:

• Sample preparation: For placental and bone marrow samples, use collagenase digestion (1-2 mg/ml, type IV) for 30-45 minutes at 37°C, followed by gentle mechanical dissociation to maintain cellular integrity while releasing stem cells from their niches .

• Antibody selection: Choose directly conjugated anti-FZD9 antibodies when available, as they minimize washing steps and reduce cell loss. If using unconjugated primary antibodies, select secondary antibodies with minimal background in mesenchymal tissues.

• Staining protocol:

  • Wash cells in PBS containing 2% FBS and 2mM EDTA

  • Block Fc receptors using 10% normal serum for 15 minutes

  • Stain with anti-FZD9 antibody at 1-5 μg per million cells

  • For multicolor analysis, include antibodies against CD9, CD63, CD90, CD10, CD13, and CD26

  • Incubate for 30 minutes at 4°C protected from light

  • Wash twice before analysis

• Sorting parameters: Use a low-pressure sorting setup (20-25 psi) with a 100μm nozzle to maintain viability of the larger mesenchymal stem cells. Include viability dyes (such as DAPI or 7-AAD) to exclude dead cells.

• Post-sort validation: Confirm stemness properties of isolated FZD9+ cells through:

  • Colony-forming unit-fibroblastic (CFU-F) assays

  • Expression analysis of stemness markers (Oct-4, nanog, SSEA-4)

  • Differentiation assays into adipogenic and osteogenic lineages

Research has demonstrated that FZD9+ cells from placental tissues show approximately 60-fold enrichment for CFU-F when compared to unsorted populations, with clonogenic cells residing exclusively in the FZD9+ fraction .

How do different tissue fixation methods affect FZD9 epitope detection?

The selection of fixation method significantly impacts FZD9 epitope preservation and accessibility. Based on systematic comparisons:

When studying FZD9 in specialized contexts like neuronal tissue or embryonic structures, carefully balance fixation intensity against epitope preservation based on the specific antibody's requirements.

What approaches can distinguish the roles of FZD9 in normal development versus pathological conditions?

To differentiate between developmental and pathological functions of FZD9, researchers should consider these methodological approaches:

• Conditional knockout models: Unlike global Fzd9-/- mice, which develop abnormal B-cell phenotypes with age (splenomegaly, thymic atrophy, lymphadenopathy) , tissue-specific or inducible knockouts allow precise temporal control for distinguishing developmental from homeostatic functions.

• Developmental timing analysis: Use FZD9 antibodies to track expression patterns across developmental stages, particularly in B-cell development where FZD9 plays a critical role at the pre-B cell stage during clonal expansion .

• Pathway inhibition studies: Compare the effects of FZD9 neutralizing antibodies with pharmacological Wnt pathway inhibitors (like C59) to distinguish receptor-specific versus pathway-general effects. Research in pancreatic islet models demonstrated that Wnt inhibition largely recapitulates the phenotypes observed in Fzd9-deficient mice .

• Competitive reconstitution assays: In hematopoietic research, competitive bone marrow transplantation using mixed wild-type and Fzd9-/- cells allows quantification of cell-intrinsic versus microenvironment-dependent defects. This approach revealed that the pre-B cell developmental defect in Fzd9-/- mice is partially intrinsic to the hematopoietic system .

• Disease model comparison: Apply FZD9 antibodies across normal tissues, developmental samples, and pathological specimens to identify disease-specific alterations in expression patterns or subcellular localization.

When interpreting results, consider that FZD9 functions may vary dramatically between different cellular contexts. For example, while FZD9 supports B-cell development in normal hematopoiesis, it may contribute to oncogenesis in epithelial tissues through distinct molecular mechanisms.

How can researchers optimize Western blotting protocols for detecting FZD9?

Western blotting for FZD9 presents unique challenges due to its transmembrane nature and potential post-translational modifications. For optimal results:

• Sample preparation: Use specialized lysis buffers containing 1-2% non-ionic detergents (such as Triton X-100 or NP-40) supplemented with protease inhibitors to extract membrane-bound FZD9 efficiently.

• Protein denaturation: Heat samples at 70°C rather than boiling (95-100°C) to prevent aggregation of transmembrane domains. For studying non-denatured complexes, consider blue native PAGE techniques.

• Gel selection: Use gradient gels (4-12% or 4-15%) to accommodate both monomeric FZD9 (~60 kDa) and potential higher molecular weight complexes or glycosylated forms.

• Transfer conditions: Employ semi-dry transfer systems with 20% methanol for optimal transfer of hydrophobic proteins. For larger complexes, wet transfer with reduced methanol (10%) may improve efficiency.

• Blocking and antibody incubation: Use 5% non-fat milk in TBST for blocking, but switch to 3-5% BSA for antibody incubations, as milk proteins can sometimes interfere with detection of certain epitopes on transmembrane proteins.

• Controls and validation: Include positive controls (tissues with high FZD9 expression like brain extracts) and negative controls (FZD9 knockout tissue or cells). For validation of band specificity, consider using multiple antibodies targeting different epitopes of FZD9 .

When troubleshooting faint or absent signals, consider enriching for membrane fractions through ultracentrifugation or using biotinylation techniques to specifically isolate and concentrate cell surface proteins before Western blot analysis.

What are the primary considerations when using FZD9 antibodies for chromatin immunoprecipitation (ChIP) studies?

For researchers exploring transcriptional regulation of FZD9 or its downstream targets through ChIP:

• Crosslinking optimization: Standard 1% formaldehyde for 10 minutes may be insufficient for capturing transient interactions. Consider dual crosslinking with 2mM disuccinimidyl glutarate (DSG) for 30 minutes followed by 1% formaldehyde for 10 minutes to stabilize multi-protein complexes.

• Chromatin fragmentation: Aim for fragments of 200-500bp through careful optimization of sonication parameters for each cell type or tissue. Excessive sonication can destroy epitopes, while insufficient fragmentation reduces resolution.

• Antibody selection: Choose ChIP-validated antibodies specifically proven to immunoprecipitate FZD9 or its associated transcription factors. For studying Myc-FZD9 interactions, antibodies against Myc have been successfully used to demonstrate direct binding to the FZD9 promoter .

• Controls:

  • Input chromatin (pre-immunoprecipitation sample)

  • IgG control (matching the host species of the primary antibody)

  • Positive control (antibody against a known abundant transcription factor)

  • Negative control regions (gene deserts or repressed genes)

• Validation approaches:

  • Perform ChIP-qPCR on known target regions before proceeding to genome-wide methods

  • Use multiple antibodies targeting different epitopes of the same protein

  • Confirm findings in knockout/knockdown systems

Research has demonstrated that FZD9 is a direct transcriptional target of Myc, which has been validated through ChIP analysis showing Myc binding to the FZD9 promoter region .

How can researchers distinguish between different frizzled family members when using antibodies?

Cross-reactivity between frizzled family members represents a significant challenge in FZD9 research. To ensure specificity:

• Epitope selection: Choose antibodies raised against the least conserved regions of FZD9, typically the intracellular C-terminal domain or the variable regions of the extracellular N-terminal domain.

• Validation in knockout systems: Test antibodies on samples from Fzd9-/- mice or FZD9-knockdown cell lines to confirm absence of signal. This is particularly important when studying tissues that may express multiple frizzled family members .

• Peptide competition: Perform pre-absorption with specific blocking peptides corresponding to the immunogen used to generate the antibody. Compare staining patterns before and after peptide blocking to identify non-specific signals.

• Multi-antibody approach: Use multiple antibodies targeting different epitopes of FZD9 and confirm consistent patterns of expression and localization.

• Expression profiling validation: Correlate protein detection with mRNA expression data (qPCR or RNA-seq) to confirm that detected signals match expected expression patterns across tissues or experimental conditions.

• Cross-reactivity testing: When possible, test antibodies against recombinant proteins of other frizzled family members, particularly FZD3, which has been noted as an alias for FZD9 in some contexts and may share structural similarities .

For applications requiring absolute specificity, consider complementing antibody-based approaches with genetic tagging methods or reporter systems when feasible.

How are FZD9 antibodies being utilized in stem cell research beyond traditional applications?

FZD9 antibodies have emerged as powerful tools for identifying and isolating progenitor populations with specific differentiation potential:

• Prospective isolation: Beyond established applications in mesenchymal stem cell isolation , researchers are using FZD9 antibodies to identify novel progenitor populations in diverse tissues. For example, Fzd9+ supporting cells have been identified as progenitors for certain specialized cell types .

• Lineage tracing: By combining FZD9 antibodies with EdU labeling techniques, researchers can track the proliferation and differentiation of specific progenitor populations. Studies have demonstrated that isolated Fzd9+ cells generate similar numbers of specialized cells compared to other established progenitor populations (e.g., Lgr5+ cells) in differentiation assays .

• Regenerative medicine applications: FZD9 antibodies enable the isolation of purified progenitor populations for therapeutic applications. The demonstration that Fzd9+ cells express embryonic stem cell markers (Oct-4, nanog, SSEA-4) while maintaining multi-lineage differentiation potential makes them promising candidates for regenerative medicine approaches .

• Single-cell analysis: Combining FZD9 antibody-based sorting with single-cell transcriptomics allows high-resolution characterization of progenitor heterogeneity and developmental trajectories. This approach has revealed that FZD9+ populations may contain distinct subpopulations with varying differentiation potentials.

• Co-expression profiling: Multi-parameter analysis using FZD9 antibodies in combination with other markers (such as CD10, CD26) has identified highly enriched progenitor populations with enhanced colony-forming efficiency (60-fold enrichment in the FZD9+CD10+CD26+ fraction) .

These applications highlight the evolving utility of FZD9 antibodies beyond classical receptor characterization, positioning them as valuable tools in stem cell biology and regenerative medicine research.

What methodological approaches can elucidate FZD9's role in the pathogenesis of Williams-Beuren syndrome?

Williams-Beuren syndrome (WBS) involves heterozygous deletion of the chromosome region containing FZD9, yet understanding FZD9's specific contribution requires sophisticated methodological approaches:

• Genotype-phenotype correlation: Use FZD9 antibodies to quantify protein expression levels in patient-derived samples, correlating expression with specific clinical manifestations of WBS. This approach may help distinguish FZD9-specific effects from those caused by deletion of other genes in the region.

• Patient-derived cellular models: Apply FZD9 antibodies in immunofluorescence and biochemical studies of patient-derived cells to characterize alterations in Wnt signaling dynamics and downstream pathway activation.

• CRISPR-based rescue models: In WBS patient-derived cells or animal models, use CRISPR-mediated rescue of FZD9 expression followed by antibody-based validation to determine which phenotypes can be specifically attributed to FZD9 haploinsufficiency.

• Comparative studies with Fzd9-/- mice: While Fzd9-/- mice do not recapitulate obvious features of WBS, they do exhibit specific B-cell developmental abnormalities . Use FZD9 antibodies to characterize lymphoid development in WBS patients to determine if similar hematopoietic phenotypes exist.

• Conditional knockout approaches: Generate tissue-specific or developmental stage-specific Fzd9 knockout models using Cre-lox systems, then use FZD9 antibodies to confirm deletion patterns and correlate with phenotypic outcomes.

These methodological approaches can help distinguish the specific contributions of FZD9 haploinsufficiency to WBS pathogenesis from effects caused by other deleted genes in the 7q11.23 region.

How should researchers interpret changes in FZD9 expression patterns in disease states?

Interpreting alterations in FZD9 expression requires careful consideration of several methodological and biological factors:

• Context-dependent functions: FZD9 exhibits both tumor-promoting and tumor-suppressive activities depending on cellular context . When analyzing expression changes, consider:

  • Tissue-specific baseline expression levels

  • Concurrent alterations in Wnt ligands and downstream effectors

  • Changes in other frizzled family members that may compensate for or synergize with FZD9

• Quantification approaches: When using antibody-based detection methods to measure FZD9 expression changes:

  • Employ multiple detection techniques (IHC, Western blot, flow cytometry)

  • Use consistent analysis parameters across samples

  • Include gradient controls to validate expression level comparisons

  • Normalize to appropriate housekeeping proteins or reference genes

• Subcellular localization: Beyond simple expression levels, examine changes in:

  • Membrane versus cytoplasmic localization

  • Nuclear translocation patterns

  • Association with lipid rafts or signaling endosomes

• Functional validation: Correlate expression changes with functional outcomes using:

  • Cell proliferation assays

  • Colony formation studies

  • Migration and invasion assays

  • Stem cell function (for FZD9+ progenitor populations)

• Clinical correlation: For translational studies, validate findings through:

  • Tissue microarray analysis with clinical outcome correlation

  • Comparison across disease stages or grades

  • Multivariate analysis to control for confounding factors

The dual nature of FZD9 as both an oncogene and tumor suppressor highlights the importance of comprehensive analysis beyond simple expression level changes to understand its functional significance in disease states.

What statistical methods are most appropriate for analyzing FZD9 antibody-generated data in heterogeneous tissues?

When analyzing FZD9 expression in complex tissues containing multiple cell types:

• Single-cell analysis approaches:

  • Flow cytometry with multi-parameter gating strategies to identify specific subpopulations

  • Single-cell RNA-seq correlation with protein expression data

  • Imaging mass cytometry for spatial resolution of expression patterns

• Quantitative image analysis for immunohistochemistry/immunofluorescence:

  • Cell-type specific masking using co-staining with lineage markers

  • Automated tissue segmentation algorithms

  • Nearest-neighbor analysis for spatial relationships

• Statistical considerations:

  • Non-parametric tests for non-normally distributed expression data

  • Mixed effects models to account for inter-individual and inter-sample variation

  • Multiple testing correction for high-dimensional datasets

  • Bootstrapping approaches for small sample sizes

• Validation strategies:

  • Technical replicates to assess method variability

  • Biological replicates to assess population heterogeneity

  • Independent cohort validation for clinical studies

  • Cross-platform validation (protein vs. mRNA detection)

• Visualization approaches:

  • Dimensionality reduction techniques (t-SNE, UMAP) for multi-parameter data

  • Spatial heatmaps for tissue section analysis

  • Violin plots to visualize distribution patterns within populations

For studies examining FZD9+ cells in heterogeneous tissues such as bone marrow or placenta, these approaches have enabled the identification of rare progenitor populations (approximately 0.2% of total cells) with specific functional properties .