PAX8 Antibody

What is PAX8 and why is it important in scientific research?

PAX8 is a member of the paired box (PAX) family of transcription factors that plays crucial roles in embryonic development, particularly in the thyroid and urogenital tract. It functions as a transcription factor for thyroid-specific gene expression, maintaining the functional differentiation of thyroid cells . PAX8 is essential for organogenesis during embryonic development of the kidney, thyroid, and paramesonephric ducts, which give rise to various urogenital organs including the seminal vesicles, vas deferens, ureter, uterus, and fallopian tubes .

Its sustained expression in mature tissues including normal kidney, thyroid, breast, seminal vesicles, and gynecological tissues suggests it plays a role in homeostatic control beyond development . PAX8's restricted expression pattern makes it valuable as a sensitive and specific marker for both primary and metastatic tumors derived from these tissues, making PAX8 antibodies invaluable tools in cancer research and diagnostics.

What are the common applications for PAX8 antibodies in research?

PAX8 antibodies are versatile research tools with multiple applications:

• Western blotting (WB): For detecting and quantifying PAX8 protein expression in tissue or cell lysates

• Immunohistochemistry (IHC-P): For visualizing PAX8 expression in formalin-fixed, paraffin-embedded tissue sections

• Immunocytochemistry/Immunofluorescence (ICC/IF): For cellular localization studies of PAX8

• Chromatin Immunoprecipitation (ChIP): For investigating PAX8 binding to DNA and its role in transcriptional regulation

• Immunoprecipitation (IP): For isolating PAX8 protein complexes to study protein-protein interactions

PAX8 antibodies exhibit nuclear localization in immunohistochemical staining, with minimal cytoplasmic expression, consistent with its role as a transcription factor .

How should I validate a PAX8 antibody before using it in my experiments?

Proper validation of PAX8 antibodies is essential for reliable results:

• Positive controls: Include tissues known to express PAX8 (thyroid gland, kidney) in your validation. Thyroid epithelial cells around follicles show strong nuclear staining when using properly validated PAX8 antibodies .

• Negative controls: Include tissues known not to express PAX8, such as most mesenchymal tissues.

• Confirm expected molecular weight: In Western blot, PAX8 should appear at approximately 48 kDa, though isoforms may exist .

• Validate specificity: Consider potential cross-reactivity issues, particularly with other PAX family members. The N-terminal region of PAX8 shows approximately 70% sequence homology with PAX5, which has led to cross-reactivity issues with some antibodies .

• Multiple detection methods: When possible, confirm expression using more than one technique (e.g., IHC and Western blot).

• Literature comparison: Compare your results with published literature on PAX8 expression patterns.

What is the significance of the PAX8-AS1/miR-96-5p/PKN2 axis in cancer research?

The PAX8-AS1/miR-96-5p/PKN2 axis represents an important regulatory pathway in papillary thyroid carcinoma (PTC). PAX8-AS1 is a long non-coding RNA (lncRNA) that functions as a tumor suppressor in PTC . Research has revealed that:

• PAX8-AS1 is downregulated in PTC tissues and cell lines compared to normal thyroid tissues and cells .

• PAX8-AS1 expression levels correlate with tumor stage, with lower expression in advanced stages (III and IV) compared to earlier stages (I and II) .

• Mechanistically, PAX8-AS1 binds to miR-96-5p, functioning as a competing endogenous RNA (ceRNA) .

• This binding inhibits miR-96-5p's suppression of PKN2, a downstream target .

• Overexpression of PAX8-AS1 inhibits PTC cell proliferation and promotes apoptosis through this pathway .

The discovery of this axis provides potential therapeutic targets for PTC treatment. Researchers investigating PAX8 antibodies should consider this regulatory mechanism when studying PAX8's role in thyroid cancer, as it may inform experimental design and interpretation of results related to PAX8 function in cancer progression.

How do I troubleshoot cross-reactivity issues between PAX8 and PAX5 antibodies?

The cross-reactivity between PAX8 and PAX5 antibodies is a significant concern in research, particularly when studying lymphoid tissues:

• Understand the source of cross-reactivity: There is substantial sequence homology (approximately 70%) in the N-terminus of PAX8 and PAX5 proteins . Antibodies raised against this region may recognize both proteins.

• Selection of appropriate antibody: Choose antibodies targeting regions with less homology between PAX family members. Monoclonal antibodies targeting specific epitopes may offer greater specificity than polyclonal antibodies targeting the N-terminal region.

• Experimental validation:

  • Use known PAX5-positive/PAX8-negative tissues (lymphoid tissues) and PAX8-positive/PAX5-negative tissues (thyroid) as controls

  • Perform knockdown/knockout experiments to confirm specificity

  • Consider using multiple antibodies targeting different epitopes of the same protein

• Data interpretation caution: Previous research suggesting PAX8 expression in lymphoid malignancies should be re-evaluated due to potential cross-reactivity with PAX5 .

• Confirmation with molecular techniques: Supplement immunohistochemical findings with RNA expression data (RT-PCR) or proteomics approaches.

When reporting research findings involving PAX8 in lymphoid tissues, explicitly address the potential for cross-reactivity and detail the validation steps taken to ensure antibody specificity.

What experimental considerations are important when investigating PAX8's role in cancer progression?

When designing experiments to investigate PAX8's role in cancer:

• Cell line selection: Choose appropriate models based on known PAX8 expression. For PTC research, cell lines like K1, TPC-1, and IHH4 have been used, with normal thyroid cell line Nthy-ori3-1 serving as control .

• Expression manipulation strategies:

  • Overexpression: Use plasmid systems like pcDNA3.1/PAX8-AS1 for gain-of-function studies

  • Knockdown/silencing: Consider shRNA or siRNA approaches, verified by qRT-PCR for efficiency

• Functional assays:

  • Proliferation: MTT assay and EdU incorporation for measuring cell proliferation

  • Apoptosis: Flow cytometry and expression of apoptotic markers (cleaved caspase 3, Bax, Bcl-2)

  • Migration/invasion: Wound healing and transwell assays

  • Colony formation assays

• Regulatory mechanism investigations:

  • For PAX8-AS1 studies: Luciferase reporter assays to verify binding relationships

  • Chromatin immunoprecipitation (ChIP) to identify PAX8 binding sites

  • Co-immunoprecipitation to identify protein interaction partners

• Downstream target analysis: Investigate expression of known PAX8 target genes using qRT-PCR or Western blot.

• Clinical correlation: Analyze PAX8 expression in patient samples in relation to clinical parameters and outcomes.

By comprehensively addressing these experimental considerations, researchers can generate more robust and clinically relevant findings regarding PAX8's role in cancer progression.

What are the optimal conditions for immunohistochemical detection of PAX8?

Optimizing immunohistochemical detection of PAX8 requires careful attention to several parameters:

• Tissue preparation:

  • Proper fixation in 10% neutral buffered formalin (10-24 hours)

  • Paraffin embedding and sectioning at 4-5 μm thickness

• Antigen retrieval:

  • Heat-induced epitope retrieval (HIER) using citrate buffer pH 6.0 is recommended

  • Steam or pressure cooker methods are effective for PAX8 antibodies

• Antibody selection and dilution:

  • Rabbit monoclonal antibodies (like EP331) show excellent specificity

  • Typical dilution ranges: 1:100 to 1:200 for monoclonal antibodies

  • Polyclonal antibodies may be used at concentrations around 2-3 μg/ml

• Detection system:

  • HRP-based detection systems work well for PAX8 IHC

  • DAB (3,3'-diaminobenzidine) provides good visualization of the nuclear signal

• Controls:

  • Positive tissue controls: Thyroid gland (epithelial cells around follicles)

  • Kidney tissue (tubular epithelium) can also serve as a positive control

  • Include negative controls (omission of primary antibody)

• Signal evaluation:

  • PAX8 shows nuclear localization with minimal cytoplasmic staining

  • Assess both staining intensity and percentage of positive cells

Following these guidelines should result in specific nuclear staining in PAX8-expressing tissues while minimizing background and non-specific staining.

What are the key considerations for Western blot analysis of PAX8?

For optimal Western blot detection of PAX8 protein:

• Sample preparation:

  • RIPA buffer is suitable for extracting PAX8 from tissues and cells

  • Include protease inhibitors to prevent degradation

  • Typical protein loading: 30-35 μg per lane

• Gel electrophoresis:

  • 10% SDS-PAGE gels are appropriate for resolving PAX8 (48 kDa)

  • Include molecular weight markers to confirm the target band size

• Transfer conditions:

  • Standard transfer protocols are effective for PAX8

  • PVDF or nitrocellulose membranes are both suitable

• Antibody selection and dilution:

  • Polyclonal antibodies: Typically used at 1 μg/mL

  • Monoclonal antibodies: Can be used at higher dilutions (1:10,000)

• Detection:

  • Chemiluminescence detection systems provide good sensitivity

  • Consider enhanced chemiluminescence for weak signals

• Controls:

  • Positive controls: Kidney tissue lysate or specific cell lines known to express PAX8

  • Loading control: Use housekeeping proteins (β-actin, GAPDH) to normalize expression

• Expected results:

  • PAX8 typically appears at 48 kDa

  • Multiple bands may represent different isoforms

  • Verify specificity using knockout/knockdown samples when possible

Optimizing these parameters should yield specific and reproducible detection of PAX8 protein in Western blot analysis.

How can ChIP assays be optimized for studying PAX8 binding to DNA?

Chromatin Immunoprecipitation (ChIP) for PAX8 requires careful optimization:

• Cell/tissue preparation:

  • Use fresh cells/tissues when possible

  • Crosslink with 1% formaldehyde for 10-15 minutes at room temperature

  • For PAX8, HEK-293T cells have been successfully used in ChIP experiments

• Chromatin shearing:

  • Optimize sonication conditions to generate DNA fragments of 200-500 bp

  • Verify shearing efficiency by agarose gel electrophoresis

• Antibody selection:

  • Choose ChIP-validated PAX8 antibodies

  • Use 5 μg of antibody per ChIP reaction as a starting point

  • Include appropriate negative controls (IgG from same species as primary antibody)

• Immunoprecipitation:

  • Pre-clear chromatin with protein A/G beads

  • Incubate chromatin with antibody overnight at 4°C

  • Collect immune complexes with protein A/G beads

• Washing and elution:

  • Use stringent washing conditions to reduce background

  • Elute DNA-protein complexes and reverse crosslinks

• Analysis methods:

  • PCR with specific primers targeting known or predicted PAX8 binding sites

  • For PAX8, primers targeting E2F1 promoter have been successfully used

  • Consider ChIP-seq for genome-wide binding site identification

• Data interpretation:

  • Compare enrichment to input chromatin and IgG control

  • Normalize to a non-target region to account for background

By carefully optimizing these parameters, researchers can effectively study PAX8 binding to target gene promoters and enhancers, providing insights into its transcriptional regulatory activities.

How should PAX8 expression patterns be interpreted in cancer diagnosis?

Interpreting PAX8 expression in cancer diagnosis requires understanding tissue-specific patterns:

• Renal cell carcinoma (RCC):

  • PAX8 is expressed in clear cell, papillary, and chromophobe RCC subtypes

  • Nuclear staining pattern is expected

  • Useful for distinguishing RCC from other cancers in metastatic settings

• Gynecological malignancies:

  • Strong expression in endometrioid carcinoma and serous carcinoma

  • Helpful in distinguishing gynecological origin in metastatic tumors

  • Consider expression levels in correlation with other markers

• Thyroid carcinomas:

  • Strong nuclear expression in most thyroid carcinomas

  • Used in conjunction with TTF-1 and thyroglobulin for thyroid origin confirmation

  • Can help differentiate anaplastic thyroid carcinoma from other high-grade malignancies

• Interpretation challenges:

  • Intensity scoring: Develop consistent scoring systems (negative, weak, moderate, strong)

  • Heterogeneity considerations: Record percentage of positive cells and distribution patterns

  • Background/non-specific staining: Distinguish from true positive staining

• Diagnostic pitfalls:

  • Beware of lymphoid tissue interpretation due to PAX5 cross-reactivity

  • Confirm unusual expression patterns with additional markers

  • Consider technical factors that may affect staining quality

A comprehensive interpretation should include assessment of staining intensity, percentage of positive cells, subcellular localization, and correlation with morphological features and other immunohistochemical markers.

What is the significance of PAX8-AS1 downregulation in cancer and how can it be studied?

PAX8-AS1, a long non-coding RNA associated with PAX8, shows significant downregulation in papillary thyroid carcinoma with important functional consequences:

• Expression patterns and clinical significance:

  • PAX8-AS1 is downregulated in PTC tissues compared to normal tissues

  • Lower expression correlates with advanced tumor stages (III and IV)

  • May serve as a potential prognostic biomarker in PTC

• Functional impact:

  • Acts as a tumor suppressor in PTC

  • Overexpression inhibits proliferation and promotes apoptosis of PTC cells

  • Functions through the miR-96-5p/PKN2 axis

• Experimental approaches to study PAX8-AS1:

  • Expression analysis: qRT-PCR for quantification in tissues and cell lines

  • Gain-of-function studies: Overexpression using pcDNA3.1/PAX8-AS1 plasmid

  • Functional assays: MTT, EdU, and flow cytometry to assess effects on proliferation and apoptosis

  • Mechanistic studies: Luciferase reporter assay to confirm binding relationships with miR-96-5p

• Data interpretation considerations:

  • Compare expression with clinicopathological parameters

  • Assess correlation with PAX8 protein expression

  • Consider the broader lncRNA-miRNA-mRNA regulatory network

• Translational potential:

  • Biomarker development for prognosis prediction

  • Potential therapeutic target for restoring normal PAX8-AS1 expression

This multilevel approach to studying PAX8-AS1 can provide comprehensive insights into its role in cancer biology and potential clinical applications.

What are the latest advances in using PAX8 antibodies for research and diagnostics?

Recent advances in PAX8 antibody applications include:

• Improved antibody specificity:

  • Development of monoclonal antibodies with enhanced specificity (e.g., EP331)

  • Recognition of the cross-reactivity issue with PAX5 and development of antibodies that minimize this problem

  • Targeting of specific epitopes to avoid non-specific binding

• Multiplexed detection systems:

  • Integration with multiplex immunohistochemistry panels

  • Use in conjunction with other cancer-specific markers for more precise tumor classification

  • Application in digital pathology and image analysis algorithms

• Enhanced detection technologies:

  • Improved sensitivity through signal amplification systems

  • Development of PAX8 antibodies compatible with chromogenic and fluorescent detection

  • Applications in automated immunostaining platforms

• Expanded diagnostic applications:

  • Refinement of PAX8's role in diagnosing tumors of unknown primary

  • Utilization in liquid biopsy approaches for circulating tumor cell identification

  • Integration into molecular diagnostic algorithms

• Research applications:

  • Study of PAX8's role in cellular reprogramming and differentiation

  • Investigation of PAX8-regulated pathways in development and disease

  • Exploration of PAX8-AS1 and other PAX8-associated non-coding RNAs

These advances continue to expand the utility of PAX8 antibodies in both basic research and clinical diagnostic applications, providing researchers with more powerful and specific tools for investigating PAX8's diverse biological functions.

How can I optimize antibody dilution for maximum signal-to-noise ratio in PAX8 detection?

Optimizing PAX8 antibody dilution requires a systematic approach:

• Titration strategy:

  • Begin with the manufacturer's recommended dilution range (e.g., 1:100 - 1:200 for monoclonal antibodies)

  • Prepare a series of dilutions (e.g., 1:50, 1:100, 1:200, 1:400)

  • Use consistent positive control tissue (thyroid or kidney) for all dilutions

• Signal evaluation parameters:

  • Assess intensity of nuclear staining in known positive cells

  • Evaluate background staining in stromal/negative cells

  • Calculate signal-to-noise ratio at each dilution

• Optimization considerations:

  • Different detection systems may require different optimal dilutions

  • Longer incubation times may allow for more dilute antibody concentration

  • Fresh vs. archival tissues may require different antibody concentrations

• Documentation:

  • Photograph results at each dilution

  • Create a dilution curve plotting signal intensity vs. antibody concentration

  • Determine the inflection point where additional antibody no longer increases specific signal

• Validation:

  • Test optimal dilution across multiple samples/tissue types

  • Verify reproducibility between batches

  • Consider lot-to-lot variations in antibody concentration

This systematic approach will help identify the optimal antibody dilution that maximizes specific signal while minimizing background, ensuring reliable and reproducible PAX8 detection.

What are the best practices for double immunostaining involving PAX8 and other markers?

Double immunostaining with PAX8 and other markers requires careful consideration of several factors:

• Marker selection and compatibility:

  • Choose markers with different cellular localizations when possible (e.g., PAX8 [nuclear] with membrane or cytoplasmic markers)

  • Consider markers expressed in different cell populations for clear distinction

  • Verify that primary antibodies are raised in different host species to avoid cross-reactivity

• Protocol optimization:

  • Sequential vs. simultaneous incubation: Sequential often yields cleaner results for PAX8

  • Antigen retrieval optimization: Select a method compatible with both markers

  • Blocking steps: Include additional blocking between sequential detections

• Detection system selection:

  • For chromogenic detection: Use contrasting colors (e.g., DAB [brown] for PAX8 and Fast Red for second marker)

  • For fluorescent detection: Choose fluorophores with minimal spectral overlap

  • Consider signal amplification for weaker markers

• Controls:

  • Single staining controls to verify individual antibody performance

  • Omission controls to assess non-specific binding of secondary antibodies

  • Tissue controls known to express both markers of interest

• Troubleshooting common issues:

  • Cross-reactivity: Use highly cross-adsorbed secondary antibodies

  • Signal bleed-through: Optimize detection system and imaging parameters

  • Uneven staining: Ensure adequate tissue permeabilization and reagent access

• Analysis and interpretation:

  • Document co-expression patterns quantitatively

  • Consider digital image analysis for objective quantification

  • Assess subcellular localization of both markers

These best practices will help ensure reliable and interpretable results when performing double immunostaining involving PAX8 and other diagnostic or research markers.