FZD9 Antibody, FITC conjugated

What are the primary research applications for FZD9 Antibody, FITC conjugated?

FZD9 Antibody, FITC conjugated is primarily used in several key research applications:

• ELISA: Typically at dilutions of 1:500-1:1000, as recommended for most commercially available antibodies .

• Flow Cytometry: Particularly useful for identifying and sorting FZD9-expressing cells in developmental and cancer studies. Optimal dilutions should be experimentally determined based on cell type and expression level .

• Immunofluorescence: The FITC conjugation enables direct visualization without secondary antibody in fluorescent microscopy at dilutions typically ranging from 1:400-1:1600 .

• Functional Assays: Used to investigate Wnt pathway activation or inhibition through FZD9 in various cell types .

In which tissues and developmental contexts is FZD9 expression most significant?

FZD9 shows tissue-specific expression patterns that are developmentally regulated:

• Neural Tissues: Highly expressed in both developing and adult hippocampus, where it marks neural stem cells .

• Cochlear Tissues: Expressed in inner phalangeal cells (IPhCs), inner border cells (IBCs), and the third-row Deiters' cells (DCs) in the postnatal cochlea, but not in inner pillar cells (IPCs) or greater epithelial ridge (GER) cells .

• Hematopoietic System: Expressed throughout B-cell development with increased expression in Hardy fractions B to C, consistent with its role in B-cell development .

• Other Tissues: Detected in testis, eye, skeletal muscle, and kidney .

Research using lineage tracing with Fzd9-CreER/Rosa26-tdTomato mice has definitively mapped FZD9 expression in the cochlea, showing that Fzd9+ cells represent a population of hair cell progenitors .

How does FZD9 function molecularly in the Wnt signaling pathway?

FZD9 functions as a receptor for Wnt proteins with several key molecular activities:

• Canonical Pathway Activation: Couples with specific Wnt ligands (notably Wnt9a) to activate the β-catenin canonical signaling pathway, leading to:

  • Activation of disheveled proteins

  • Inhibition of GSK-3 kinase

  • Nuclear accumulation of β-catenin

  • Activation of Wnt target genes

• Signaling Specificity: Research has demonstrated that FZD9 shows ligand specificity, particularly for Wnt9a. This specificity appears mediated by both the receptor's extracellular domain and intracellular regions, as shown in chimeric receptor studies .

• Pathway Cross-talk: Evidence suggests that EGFR may form a complex with Wnt9a and FZD9, providing a mechanism for signaling specificity and cross-talk between pathways .

• Non-canonical Signaling: FZD9 may also engage in non-canonical Wnt signaling involving PKC and calcium fluxes, particularly important in bone formation through ISG15-mediated processes .

The interaction specificity between Wnt9a and FZD9 has been confirmed using Super-TOP-Flash (STF) reporter assays, where this specific ligand-receptor pair showed synergistic activation .

What methodological considerations are important when using FZD9 Antibody, FITC conjugated for flow cytometry?

When using FZD9 Antibody, FITC conjugated for flow cytometry, researchers should consider:

Sample Preparation:

• Single Cell Suspension: For tissues like cochlea, use trypsin digestion methods as described in studies with Fzd9-CreER/Rosa26-tdTomato mice .

• Fixation Protocol: Light fixation (typically 2-4% PFA for 10-15 minutes) preserves epitope accessibility while maintaining cellular integrity.

Controls and Validation:

• Negative Controls: Include unstained cells and isotype control antibodies conjugated to FITC.

• Positive Controls: Use cell lines known to express FZD9 (e.g., HeLa, HUVEC, or A549 cells) .

• Blocking: Pre-incubate cells with FcR blocking reagent to prevent non-specific binding.

Technical Considerations:

• Compensation: Properly compensate for spectral overlap if using multiple fluorochromes.

• Antibody Titration: Perform titration experiments to determine optimal antibody concentration (typically starting at manufacturer's recommendation of 1:100-1:400) .

• Storage: Store conjugated antibodies at 4°C in the dark to prevent photobleaching of the FITC fluorophore .

Research has successfully used FAC sorting of Fzd9+ cells from Fzd9-CreER/Rosa26-tdTomato mouse cochleae for subsequent culture and differentiation assays .

How do researchers validate the specificity of FZD9 antibodies in their experimental systems?

Multiple validation strategies should be employed:

Genetic Validation:

• Knockout Controls: Compare staining between wild-type and Fzd9 -/- tissues/cells .

• siRNA/shRNA Knockdown: Use cells with reduced FZD9 expression through RNA interference as validation controls .

Biochemical Validation:

• Western Blot: Confirm specific detection of a band at the expected molecular weight (64-70 kDa) .

• Peptide Competition: Pre-incubate antibody with immunizing peptide to demonstrate loss of specific staining .

Orthogonal Methods:

• mRNA-Protein Correlation: Compare antibody staining with mRNA expression data (e.g., RT-PCR or RNA-seq) .

• Multiple Antibodies: Use different antibodies targeting distinct epitopes of FZD9 .

Research has validated FZD9 antibodies by demonstrating their ability to detect protein on the cell surface using both immunofluorescence and flow cytometry with antibodies directed to the extracellular region between the CRD and the first transmembrane domain .

What are the optimal storage conditions for maintaining FZD9 Antibody, FITC conjugated activity?

Proper storage is critical for maintaining antibody activity:

• Short-term Storage: Keep at 4°C in light-protected vials or covered with aluminum foil. Conjugated antibodies are stable for approximately 12 months at this temperature .

• Long-term Storage: For extended storage (up to 24 months), dilute conjugates with up to 50% glycerol and store at -20°C to -80°C .

• Avoiding Freeze-Thaw Cycles: Freeze-thaw cycles compromise both enzyme activity and antibody binding. Aliquot before freezing to minimize cycles .

• Buffer Considerations:

  Common Storage Buffers	Purpose
  PBS with 0.02% sodium azide and 50% glycerol, pH 7.3	Preserves antibody structure and prevents microbial growth
  50mM Sodium Borate with 0.05% Sodium Azide	Alternative formulation for some products

Most manufacturers ship conjugated antibodies with polar packs and recommend immediate storage upon receipt at the recommended temperature .

How can FZD9 Antibody be used to study the role of this receptor in cancer models?

FZD9 has been implicated in several cancer types, and researchers use FZD9 antibodies to:

Expression Analysis:

• Immunohistochemistry: To examine FZD9 expression in cancer tissues, particularly in gliomas, breast cancer, and pancreatic insulinomas .

• Flow Cytometry: To isolate and characterize FZD9-expressing cancer cells for downstream analysis .

Functional Studies:

• Blocking Experiments: FZD9 antibodies can be used to block Wnt signaling through this receptor in functional assays .

• Genetic Models: Combined with genetic models (e.g., Fzd9 knockout in Myc-driven pancreatic insulinoma models) to understand pathway dependencies .

Prognostic Marker Evaluation:

• Research has identified FZD9 as a potential prognostic biomarker in triple-negative breast cancer, associated with low survival, tumor recurrence, and tumor grade .

Therapeutic Development:

• FZD9 antibodies help evaluate the potential of targeting this receptor therapeutically, particularly in contexts where Wnt inhibition showed significant anti-tumor effects .

Research demonstrated that Fzd9 is essential for Myc-driven tumorigenesis in pancreatic islets, where its deficiency impaired sustained tumor expansion and β-cell dedifferentiation, allowing pancreatic islets to maintain their physiological structure .

What role does FZD9 play in stem cell and progenitor cell biology?

FZD9 functions critically in multiple stem/progenitor cell contexts:

Neural Stem Cells:

• FZD9 is selectively expressed in both developing and adult hippocampus, marking neural stem cells .

• It regulates neural progenitor cell viability through the β-catenin canonical signaling pathway by negatively regulating cell cycle arrest, leading to inhibition of neuron apoptotic processes .

Cochlear Progenitors:

• FZD9+ supporting cells in the cochlea function as progenitors for hair cell generation .

• Lineage tracing using Fzd9-CreER/Rosa26-tdTomato mice demonstrated that these cells can form spheres in vitro and generate hair cells both in vivo and in vitro .

Hematopoietic Development:

• FZD9 is expressed throughout B-cell development with increased expression at specific stages (Hardy fractions B to C) .

• Fzd9-/- mice show pronounced abnormalities in B-cell development, particularly at points where B cells undergo self-renewal prior to further differentiation .

Comparative analysis showed that isolated Fzd9+ cells have similar capacity for proliferation, differentiation, and hair cell generation as the more widely studied Lgr5+ progenitors, positioning FZD9 as a more restricted marker for hair cell progenitors .

How does the interaction between Wnt9a and FZD9 contribute to specific developmental processes?

The Wnt9a-FZD9 signaling axis regulates key developmental processes:

Hematopoietic Development:

• Wnt9a specifically signals through FZD9 for the amplification of blood progenitor cells during development .

• This interaction activates β-catenin-dependent signaling, as demonstrated using Super-TOP-Flash reporter assays .

• In zebrafish and human embryonic stem cell models, disrupting either WNT9A or FZD9 expression significantly compromised the generation of hematopoietic stem/progenitor cells (HSPCs) .

Signaling Specificity:

• The specificity of Wnt9a-FZD9 interaction involves both extracellular binding and intracellular signal transduction components .

• Studies with chimeric receptors identified specific domains in FZD9 that are critical for Wnt9a signal transduction .

• Co-culture experiments demonstrated that FZD9, but not other Frizzled receptors like FZD5, could transduce the Wnt9a signal and activate reporter activity .

Molecular Complex Formation:

• Evidence suggests that EGFR forms a complex with Wnt9a and FZD9, providing a mechanism for signaling specificity .

• This complex formation appears to be evolutionarily conserved, as zebrafish cDNAs functioned in human cells .

Research using genetic non-complementation with morpholinos in zebrafish confirmed that Wnt9a and FZD9 function in the same genetic pathway for hematopoietic development .

What experimental approaches are used to study FZD9 expression patterns in tissues?

Researchers employ multiple complementary techniques:

Genetic Lineage Tracing:

• Cre-loxP System: Using Fzd9-CreER/Rosa26-tdTomato mice to permanently label Fzd9-expressing cells and their progeny upon tamoxifen administration .

• Dual Reporter Systems: Systems like Fzd9:Gal4;UAS:GFP or Fzd9:Gal4;UAS:Cre;loxP-BFP-loxP-dsRed for more sophisticated lineage analysis .

RNA Detection:

• RT-PCR: For analysis of Fzd9 mRNA expression in sorted cell populations .

• Fluorescent in situ Hybridization: To localize fzd9 mRNA in tissues, such as the lateral plate mesoderm co-expressed with endothelial markers like fli1a .

Protein Detection:

• Immunohistochemistry: Using specific antibodies against FZD9 in tissue sections, particularly effective in gliomas tissue with TE buffer pH 9.0 for antigen retrieval .

• Immunofluorescence: For high-resolution localization of FZD9 protein in cells and tissues .

• Flow Cytometry: For quantitative analysis of FZD9 expression across cell populations .

In silico Analysis:

• Microarray Data Mining: To identify differential FZD9 expression across cancer subtypes .

• Survival Analysis: Using platforms like KM Plotter, cBioPortal, bc-GenExMiner, Prognoscan, and Roc Plotter to correlate FZD9 expression with clinical outcomes .