PAX8 Antibody, FITC conjugated

What is PAX8 and why is it significant in cancer research?

PAX8 is a member of the paired box (PAX) family of transcription factors containing a paired box domain and a paired-type homeodomain. It is expressed during organogenesis of the thyroid gland, kidney, and Mullerian system . PAX8's significance in cancer research stems from its role in regulating the expression of Wilms tumor suppressor (WT1) gene, with mutations in PAX8 associated with Wilms tumor cells, thyroid and ovarian carcinomas . It serves as a useful marker in distinguishing ovarian carcinomas from mammary carcinomas, as PAX8 is expressed in a high percentage of ovarian serous, endometrioid, and clear cell carcinomas, but only rarely in primary ovarian mucinous adenocarcinomas . Additionally, PAX8 expression is reported in renal tubules, renal cell carcinoma, nephroblastoma, and seminoma .

What are the spectral characteristics of FITC-conjugated PAX8 antibodies?

FITC-conjugated PAX8 antibodies have specific excitation and emission wavelengths that define their optical properties and applications:

These spectral characteristics are important when designing multicolor immunofluorescence experiments, as researchers must ensure minimal spectral overlap between fluorophores .

What are the recommended applications and dilutions for PAX8 antibody, FITC conjugated?

PAX8 antibody, FITC conjugated, is suitable for several research applications with specific recommended dilutions:

For all applications, it is strongly recommended that the antibody be titrated in each specific testing system to obtain optimal results . Sample-dependent variations may occur, necessitating examination of validation data provided by antibody suppliers .

What is the proper storage protocol for maintaining PAX8 antibody, FITC conjugated activity?

Proper storage of FITC-conjugated PAX8 antibody is crucial for maintaining its fluorescence intensity and binding specificity:

• Temperature: Store at -20°C for long-term storage or 4°C in the dark for short-term use

• Light protection: Crucially important as FITC is photosensitive; avoid exposure to light during storage and handling

• Buffer composition: Typically stored in PBS with additives such as 50% Glycerol, 0.05% Proclin300, 0.5% BSA, pH 7.3

• Stability: Generally stable for one year after shipment when stored properly

• Aliquoting considerations: While some sources mention aliquoting is unnecessary for -20°C storage , creating small working aliquots is generally recommended practice to avoid repeated freeze-thaw cycles

For FITC-conjugated antibodies specifically, protection from light is particularly important as exposure to light accelerates photobleaching of the fluorophore, which can significantly reduce signal intensity during experiments.

How should I optimize immunofluorescence protocols with PAX8 antibody, FITC conjugated?

Optimizing immunofluorescence staining with FITC-conjugated PAX8 antibody requires attention to several critical parameters:

Fixation and Permeabilization:

• Use 4% formaldehyde fixation for 10 minutes followed by permeabilization with 0.1% Triton X-100 for 5 minutes

• For nuclear transcription factors like PAX8, efficient permeabilization is essential for antibody access

Blocking:

• Implement 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1 hour to reduce background fluorescence

• The blocking solution should match the host species of the secondary antibody if using a detection system

Antibody Dilution and Incubation:

• Begin with the recommended dilution range (1:50-1:500)

• For nuclear antigens like PAX8, overnight incubation at 4°C often yields better signal-to-noise ratios

• Include positive controls from known PAX8-expressing cells (SKOV-3 or OVCAR-3 cells are recommended)

Nuclear Counterstaining:

• DAPI is commonly used for nuclear counterstaining, which helps verify the nuclear localization of PAX8

• The nuclear counterstain should have minimal spectral overlap with FITC

Mounting Considerations:

• Use an anti-fade mounting medium specifically formulated to preserve FITC fluorescence

• Seal edges of coverslips to prevent drying and oxidation of the fluorophore

For challenging samples or when higher sensitivity is required, consider implementing a signal amplification system, though this should be carefully validated to ensure specificity is maintained.

What controls are essential when using PAX8 antibody, FITC conjugated?

Proper controls are crucial for generating reliable and interpretable data with FITC-conjugated PAX8 antibodies:

Positive Controls:

• Cell lines with known PAX8 expression: SKOV-3, OVCAR-3, or thyroid carcinoma cell lines

• Tissue sections from thyroid, kidney, or fallopian tube epithelium

• These controls validate the antibody's ability to detect the target protein

Negative Controls:

• PAX8-negative cell lines or tissues

• Isotype control antibodies (matching the primary antibody's host species and isotype) at equivalent concentration

• These controls help establish thresholds for non-specific binding

Technical Controls:

• Unstained samples to assess autofluorescence

• For flow cytometry: Fluorescence Minus One (FMO) controls to set accurate gates

• For multicolor imaging: single-color controls for spectral compensation

Specificity Validation Controls:

• Pre-absorption with immunizing peptide when available

• Parallel staining with alternative PAX8 antibodies from different clones

• Correlation with PAX8 mRNA expression data

• siRNA knockdown samples where feasible

Implementing these controls systematically ensures that the observed staining truly represents PAX8 protein rather than artifacts or non-specific binding, which is particularly important in publications and when establishing new protocols.

How can I design multicolor flow cytometry panels incorporating PAX8 antibody, FITC conjugated?

Designing effective multicolor flow cytometry panels with FITC-conjugated PAX8 antibody requires strategic planning to minimize spectral overlap and maximize information content:

Spectral Considerations:

• FITC has excitation maximum at 495 nm and emission at 519 nm

• Avoid fluorophores with significant spectral overlap such as PE or GFP

• Compatible fluorophores include APC, PE-Cy7, BV421, and PerCP-Cy5.5

Example Panel Design:

Staining Protocol Considerations:

• For nuclear antigens like PAX8, implement a sequential staining approach:

  • Surface markers first (if applicable)

  • Fixation (4% formaldehyde, 10 minutes)

  • Permeabilization (0.1% Triton X-100, 5 minutes)

  • Nuclear staining with PAX8-FITC

• Use the recommended concentration of 0.20 μg per 10^6 cells in 100 μl suspension

Essential Controls:

• Include single-color controls for compensation

• FMO controls for accurate gating

• Isotype controls at identical concentrations

• Positive and negative biological controls

Analysis Strategy:

• Gate sequentially: viable cells → single cells → target population → PAX8 expression

• Consider PAX8 expression intensity (MFI) alongside percentage of positive cells

• For heterogeneous samples, correlate PAX8 expression with other markers to identify specific cell populations

This approach enables accurate quantification of PAX8 expression in complex cellular populations and facilitates comparison between experimental conditions.

What is the relationship between PAX8 expression and SOX17 in ovarian cancer research?

Recent research has revealed important interactions between PAX8 and SOX17 in ovarian cancer, with significant implications for understanding disease mechanisms:

Physical Interaction Evidence:

• PAX8 and SOX17 physically interact as demonstrated by co-immunoprecipitation studies

• This interaction is markedly increased in high-grade serous ovarian carcinoma (HGSOC) compared to fallopian tube epithelial (FTE) cells

• Size-exclusion chromatography has shown that PAX8 and SOX17 form part of a larger complex of approximately 600 kDa

Subcellular Co-localization:

• High-resolution immunofluorescence analyses have demonstrated nuclear co-localization of PAX8 and SOX17 in:

  • Immortalized fallopian tube secretory cell lines (FT194, FT246, and FT282)

  • HGSOC cell lines (OVCAR4, KURAMOCHI, and OVSAHO)

Proximity Ligation Assay Confirmation:

• In situ proximity ligation assay (PLA) has confirmed increased interaction between PAX8 and SOX17 in HGSOC cell lines

• These protein-protein interactions are localized in the nuclei, consistent with their roles as transcription factors

Research Implications:

• FITC-conjugated PAX8 antibodies provide valuable tools for studying these interactions in multicolor imaging experiments

• The increased PAX8-SOX17 interaction in cancer cells suggests altered transcriptional regulation that may contribute to carcinogenesis

• Understanding these interactions may reveal new therapeutic targets for ovarian cancer treatment

The discovery of this interaction highlights how PAX8 antibodies contribute to mechanistic studies beyond simple protein detection, advancing our understanding of transcriptional regulation in cancer.

How can I use PAX8 antibody, FITC conjugated for proximity ligation assays (PLA) to study protein interactions?

Proximity Ligation Assay (PLA) is a powerful technique for visualizing protein-protein interactions in situ. Adapting this method for use with FITC-conjugated PAX8 antibodies requires specific methodological considerations:

Primary Antibody Selection and Preparation:

• When using PAX8 antibody, FITC conjugated for PLA:

  • Ensure the conjugation doesn't interfere with the antibody's epitope binding

  • Select an unconjugated antibody against the potential interaction partner (e.g., SOX17)

  • Verify antibodies are from different host species to enable species-specific secondary antibody recognition

PLA Probe Selection:

• Choose PLA probes that emit in spectral regions distinct from FITC (495/519 nm)

• Red or far-red emitting PLA probes (e.g., 594 nm or 647 nm) provide good spectral separation

Protocol Steps:

• Sample Preparation: Fix cells with 4% formaldehyde (10 min) and permeabilize with 0.1% Triton X-100 (5 min)

• Blocking: Use Duolink blocking solution or similar to reduce non-specific binding

• Primary Antibody Incubation: Apply both PAX8-FITC and partner antibody simultaneously or sequentially

• PLA Probe Application: Use PLA probes specific to the host species of each primary antibody

• Ligation and Amplification: Follow standard PLA protocol

• Counterstaining: DAPI for nuclear visualization

Analysis Considerations:

• The FITC signal identifies PAX8-expressing cells/regions

• PLA signals (typically appearing as distinct fluorescent dots) indicate specific interaction sites

• Quantify the number and intensity of PLA signals within FITC-positive regions

• Compare interaction frequencies between experimental conditions (e.g., normal vs. cancer cells)

This approach has been validated in studies examining PAX8-SOX17 interactions in ovarian cancer, where increased interaction frequency was observed in high-grade serous ovarian carcinoma compared to normal fallopian tube epithelial cells .

What are the key differences in PAX8 detection between normal and cancerous tissues?

Understanding PAX8 expression patterns across normal and cancerous tissues is crucial for diagnostic applications and research interpretations:

Expression Pattern Comparison:

Methodological Implications:

• Antibody Titration: Expression levels vary between tissues, requiring careful antibody titration

• Signal Intensity Assessment: Cancer tissues may show altered expression intensity requiring appropriate exposure settings

• Background Considerations: Some cancerous tissues have higher autofluorescence requiring stringent controls

• Multiplexing Strategy: Co-staining with lineage markers helps distinguish tumor cells in heterogeneous samples

Research Applications:

• PAX8-FITC antibodies facilitate identification of tumor origin in metastatic lesions

• The differential interaction between PAX8 and SOX17 in normal versus cancer cells provides insight into altered transcriptional networks

• Flow cytometric analysis with PAX8-FITC enables quantitative assessment of expression changes during malignant transformation

• Correlation of PAX8 expression with clinical outcomes may reveal prognostic significance

These differences underscore PAX8's value as both a diagnostic marker and a research tool for understanding fundamental aspects of carcinogenesis.

What troubleshooting approaches are recommended for weak or nonspecific staining with PAX8 antibody, FITC conjugated?

When encountering staining issues with FITC-conjugated PAX8 antibodies, a systematic troubleshooting approach is essential:

Addressing Weak Staining:

• Antibody Concentration Adjustment:

  • Increase antibody concentration within recommended range (1:50-1:500)

  • Perform titration experiments to determine optimal concentration for your specific sample

• Antigen Retrieval Enhancement:

  • For FFPE tissues: Optimize heat-induced epitope retrieval with citrate buffer (pH 6.0) or EDTA buffer (pH 9.0)

  • For cell lines: Test different permeabilization reagents or extend permeabilization time

• Incubation Optimization:

  • Extend primary antibody incubation (overnight at 4°C rather than 1-2 hours at room temperature)

  • Ensure consistent temperature during incubation periods

• Signal Amplification Strategies:

  • Consider signal amplification systems compatible with FITC detection

  • Verify any amplification approach maintains specificity

Resolving Nonspecific Staining:

• Blocking Protocol Enhancement:

  • Increase blocking time or concentration (1% BSA/10% normal goat serum/0.3M glycine in PBS-T for 1-2 hours)

  • Add carrier protein in antibody diluent (0.5% BSA)

  • Consider adding 5-10% serum from the host species of any secondary antibody used

• Washing Procedure Optimization:

  • Implement additional wash steps or extend washing duration

  • Ensure wash buffer contains appropriate detergent concentration (0.05-0.1% Tween-20)

• Autofluorescence Reduction:

  • Include autofluorescence quenching step (e.g., 0.1% Sudan Black in 70% ethanol)

  • For tissue sections: Consider specialized autofluorescence quenchers

• FITC-Specific Considerations:

  • Protect from light during all protocol steps to prevent photobleaching

  • Verify microscope filter sets are optimized for FITC detection

  • Check pH of buffers (FITC fluorescence is pH-sensitive)

Essential Controls for Problem Identification:

• Positive control (SKOV-3 or OVCAR-3 cells are recommended)

• Negative control (cell line known not to express PAX8)

• Isotype control at equivalent concentration

• Secondary antibody-only control (if using detection systems)

Systematic evaluation of these factors, combined with careful documentation of modifications, should resolve most staining issues with FITC-conjugated PAX8 antibodies and lead to reproducible, specific detection.

How can I quantitatively analyze PAX8 expression using FITC-conjugated antibodies in high-throughput screening?

Quantitative analysis of PAX8 expression using FITC-conjugated antibodies in high-throughput screening requires standardized approaches and rigorous controls:

Flow Cytometry-Based Quantification:

• Sample Preparation Protocol:

  • Standardize cell fixation (4% formaldehyde, 10 minutes) and permeabilization (0.1% Triton X-100, 5 minutes)

  • Use consistent antibody concentration: 0.20 μg PAX8-FITC per 10^6 cells in 100 μl suspension

  • Include calibration beads with defined fluorophore molecules for standardization

• Quantification Metrics:

  • Mean Fluorescence Intensity (MFI) of PAX8-FITC signal

  • Percentage of cells above threshold (PAX8-positive population)

  • Molecules of Equivalent Soluble Fluorochrome (MESF) for standardized cross-experimental comparison

High-Content Imaging Approaches:

• Automated Microscopy Setup:

  • Seed cells in microtiter plates (96 or 384-well format) with consistent density

  • Implement standardized staining protocol with PAX8-FITC (1:50-1:500) and nuclear counterstain

  • Acquire images using identical exposure settings across all plates

• Image Analysis Parameters:

  • Nuclear PAX8-FITC intensity (integrated or mean)

  • Nuclear area positive for PAX8 signal

  • Correlation of PAX8 expression with morphological features

  • Population heterogeneity assessment

Standardization for Screening Applications:

• Quality Control Implementation:

  • Include standard control wells on each plate (positive, negative, dynamic range)

  • Calculate Z'-factor to assess assay quality and suitability for screening

  • Monitor day-to-day variability using reference samples

• Data Normalization Strategies:

  • Percent of control normalization relative to positive and negative controls

  • Z-score normalization within plates to account for plate-to-plate variation

  • Consider robust statistical methods resistant to outliers

Application to Functional Genomics:

• Correlation of PAX8 expression changes with genetic perturbations (siRNA, CRISPR)

• Identification of compounds that modulate PAX8 expression or localization

• Integration with other cellular readouts (viability, proliferation, differentiation)

This approach enables reproducible quantification of PAX8 expression across large sample sets, facilitating the identification of factors that regulate this transcription factor in normal and disease contexts.

What considerations are important when detecting PAX8 in ovarian cancer stem cells using FITC-conjugated antibodies?

Detecting PAX8 in ovarian cancer stem cells (CSCs) presents unique challenges that require specific methodological considerations:

Cell Identification and Isolation:

• Ovarian CSCs are typically identified using markers such as CD133, CD44, ALDH activity, or sphere-forming ability

• Consider whether PAX8 expression might differ between CSC and non-CSC populations

Protocol Optimization for CSC Analysis:

• Sample Preparation:

  • Minimize processing steps that might alter stemness characteristics

  • Consider gentle fixation protocols (2% paraformaldehyde) to preserve epitopes and CSC markers

  • Optimize permeabilization to maintain both surface stemness markers and nuclear access

• Antibody Titration:

  • CSCs may express different levels of PAX8 compared to bulk tumor cells

  • Perform careful titration experiments specifically with purified CSC populations

• Multimarker Panel Design:

  Marker	Function	Suggested Fluorophore Combination
  PAX8	Transcription factor	FITC
  CD133	CSC marker	APC
  CD44	CSC marker	PE-Cy7
  ALDH	Functional CSC marker	Detected via Aldefluor (BAAA)
  Viability dye	Dead cell exclusion	Far-red fluorescent dye

Analysis Considerations:

• Flow Cytometry Approach:

  • Implement hierarchical gating: viable cells → single cells → CSC marker-positive → PAX8 analysis

  • Assess whether PAX8 expression correlates positively or negatively with stemness markers

  • Compare PAX8 expression in matched CSC and non-CSC populations

• Imaging Analysis:

  • Use confocal microscopy for precise localization of PAX8 in CSC populations

  • Implement z-stack acquisition to fully capture nuclear distribution

  • Consider intensity quantification in identifiable CSC vs. non-CSC populations

Technical Challenges and Solutions:

• CSCs often represent a small percentage of the total tumor cell population, requiring efficient enrichment

• Autofluorescence can be problematic; include unstained controls and consider autofluorescence reduction methods

• PAX8 expression might be heterogeneous within the CSC population, necessitating single-cell analysis approaches

Validation Strategies:

• Confirm PAX8 expression in sorted CSC populations using alternative methods (qPCR, western blot)

• Correlate PAX8 expression with functional stemness assays (sphere formation, tumor initiation)

• Assess PAX8 expression changes during CSC differentiation experiments

These specialized considerations will enable reliable detection and characterization of PAX8 in ovarian cancer stem cells, potentially revealing its role in stemness maintenance and therapeutic resistance.

What are the most recent advances in understanding PAX8 function revealed through antibody-based studies?

Recent antibody-based studies have significantly advanced our understanding of PAX8 biology and function in both normal and pathological contexts:

Protein Interaction Networks:

• Biochemical affinity-purification methods have identified novel PAX8-interacting proteins in ovarian carcinoma cell lines (OVCAR4, KURAMOCHI, OVSAHO) and immortalized fallopian tube secretory cells

• Size-exclusion chromatography has revealed that PAX8 exists in a complex of approximately 600 kDa

• Mass spectrometry analysis has identified SOX17 as a significant PAX8-interacting partner

Differential Interactions in Cancer:

• Co-immunoprecipitation studies have demonstrated that the level of PAX8-SOX17 complexes is markedly increased in high-grade serous ovarian carcinoma compared to fallopian tube epithelial cells

• Proximity ligation assays have confirmed increased PAX8-SOX17 interactions in HGSOC cell lines

• These findings suggest altered transcriptional regulation as a component of ovarian carcinogenesis

Subcellular Localization Insights:

• High-resolution immunofluorescence analyses have confirmed the nuclear co-localization of PAX8 and SOX17 in both normal and cancer cell lines

• The nuclear localization pattern provides important context for understanding PAX8's function as a transcription factor

Functional Implications:

• The discovery of these protein interactions suggests PAX8 participates in larger transcriptional complexes

• Different interaction patterns between normal and cancer cells may represent potential therapeutic targets

• The specific roles of these interactions in regulating gene expression programs are active areas of investigation

These advances demonstrate how antibody-based approaches continue to reveal new aspects of PAX8 biology beyond simple detection, contributing to our understanding of fundamental biological processes and disease mechanisms.

How does antibody selection impact experimental outcomes in PAX8 research?

The choice of PAX8 antibody has significant implications for experimental outcomes and data interpretation in research applications:

Clone-Specific Considerations:

• Different antibody clones may recognize distinct epitopes of PAX8, potentially affecting detection in contexts where protein interactions mask specific regions

• Monoclonal antibodies like PAX8/1491 + PAX8/1492 offer high specificity but might be sensitive to epitope modifications

• Polyclonal antibodies may detect multiple epitopes, providing more robust detection but potentially higher background

Conjugation Effects:

• FITC conjugation may affect antibody binding characteristics compared to unconjugated versions

• Direct conjugation eliminates secondary antibody steps, reducing background but potentially limiting signal amplification options

• The physical properties of the fluorophore (size, charge) can impact tissue penetration and nuclear access

Application-Specific Performance:

• An antibody performing well in flow cytometry may not be optimal for immunohistochemistry

• Fixation methods can differentially affect epitope accessibility for different antibody clones

• Sample type (fresh frozen vs. FFPE) may influence antibody selection decisions

Validation Requirements:

Research Impact Considerations:

• Discrepancies in research findings may sometimes be attributed to antibody differences

• Reproducibility challenges can arise from inconsistent antibody selection across studies

• Comprehensive reporting of antibody details (clone, manufacturer, lot, dilution) is essential for research transparency

Careful selection of the appropriate PAX8 antibody based on the specific research application, combined with rigorous validation, is crucial for generating reliable and reproducible results in both basic research and clinical studies.