PAX2 Antibody Pair

What is PAX2 and why is it important in research?

PAX2 is a member of the paired box family of transcription factors with a molecular weight of approximately 42kDa. It contains a highly conserved DNA sequence within the paired box region that encodes a DNA-binding domain, enabling PAX proteins to bind to promoters of specific genes to regulate their expression transcriptionally . PAX2 plays critical roles in the development and proliferation of the kidney, brain, and Müllerian organs, making it essential for the formation of these structures during embryogenesis . In research, PAX2 is particularly important as a marker for renal development, urogenital tract formation, and as a diagnostic tool for certain cancers including renal cell carcinomas and ovarian carcinomas . The dysregulation of PAX2 expression has been implicated in various pathological conditions, making it a valuable research target for both developmental biology and cancer research.

What tissue types normally express PAX2?

PAX2 expression follows a specific pattern across tissues:

PAX2 is specifically expressed in the developing central nervous system, eye, ear, and urogenital tract, and is essential for the development of these organs . In normal adult tissues, PAX2 is mainly detected in the urogenital system, including kidney, ureteric epithelium, fallopian tube epithelium, ovary, and uterus . Normal proliferative and secretory endometrium typically show high levels of PAX2 expression .

How do I select the appropriate PAX2 antibody for my research?

Selecting the appropriate PAX2 antibody depends on several factors, including your experimental application, target species, and whether you require monoclonal or polyclonal antibodies:

• Application compatibility: Determine which applications you need the antibody for (IHC-P, WB, ELISA, etc.). For example, the EPR8586 clone is suitable for IHC-P and WB on human samples , while other antibodies may be optimized for ELISA applications .

• Clonality considerations: Monoclonal antibodies like EPR8586 and ZR224 offer high specificity for a single epitope, making them ideal for consistent results across experiments. Polyclonal antibodies may provide higher sensitivity by recognizing multiple epitopes but potentially with more background.

• Validation status: Review published literature demonstrating the antibody's use in your specific application. For example, some PAX2 antibodies have been cited in multiple publications , providing confidence in their performance.

• Diagnostic vs. research use: Some antibodies like ZR224 are intended for in vitro diagnostic use , while others are strictly for research purposes . Ensure your selected antibody matches your intended use.

• Epitope location: Consider which region of PAX2 the antibody recognizes. Some antibodies target the C-terminal region , while others may target different epitopes.

How can PAX2 antibodies be used to differentiate between normal and pathological tissues?

PAX2 antibodies serve as valuable tools for distinguishing normal from pathological tissues, particularly in the context of renal and gynecological specimens. The pattern and intensity of PAX2 immunostaining change significantly across the spectrum from normal to neoplastic tissues:

In normal tissues, PAX2 shows consistent nuclear expression in specific cell types, particularly in the urogenital system . This provides a baseline for comparison. As tissues progress toward hyperplasia and neoplasia, characteristic changes in PAX2 expression emerge:

• Endometrial hyperplasia progression: PAX2 loss occurs early in the spectrum of endometrial hyperplasia and becomes more frequent and complete with increasing severity of lesions . Specifically:

  • Normal proliferative and secretory endometrium shows high levels of PAX2 expression

  • Simple hyperplasia exhibits variable expression

  • Complex and atypical hyperplasias show increased likelihood of complete PAX2 loss

  • FIGO grade 1 endometrioid carcinomas frequently show complete PAX2 loss

• Paired analysis advantage: When analyzing lesional tissue with adjacent normal tissue, the difference in PAX2 staining can be particularly informative. Studies have shown statistically significant differences in PAX2 expression between lesional endometrium and adjacent histologically normal endometrium across diagnoses of simple hyperplasia (p=0.011), complex hyperplasia (p<0.001), atypical hyperplasia (p<0.001), and FIGO grade 1 endometrioid cancer (p=0.003) .

• Diagnostic pattern: The consistent observation that PAX2 loss is the same or greater in lesional tissue compared to adjacent normal endometrium, with no matched adjacent normal tissue showing complete PAX2 loss, provides a reliable diagnostic pattern .

This differential expression pattern makes PAX2 antibodies valuable tools for assessment of premalignant and malignant conditions, particularly in gynecological pathology.

What are the challenges in interpreting PAX2 immunostaining, and how can they be overcome?

Interpretation of PAX2 immunostaining presents several challenges that researchers should address through methodological rigor:

• Heterogeneous expression patterns: PAX2 expression can be heterogeneous within the same sample, particularly in hyperplastic tissues . This heterogeneity necessitates careful examination of multiple fields and quantification approaches.

  Solution: Evaluate the percent of cells with PAX2 expression across multiple high-power fields. Categorizing results into defined ranges (e.g., complete loss: 0%, partial loss: 1-75%, minimal to no loss: 76-100%) can standardize interpretation .

• Background staining and false positives: Non-specific staining can complicate interpretation, particularly with polyclonal antibodies.

  Solution: Include appropriate negative controls, such as substituting primary antibody with PBS . Use monoclonal antibodies like EPR8586 or ZR224 which may provide cleaner staining with less background .

• Tissue fixation variability: Variations in fixation protocols can affect epitope preservation and antibody binding.

  Solution: Optimize antigen retrieval methods. For example, when using the EPR8586 antibody, heat-mediated antigen retrieval using Tris-EDTA buffer (pH 9.0) has been validated for consistent results .

• Distinguishing between loss patterns: Differentiating between partial and complete loss of PAX2 expression has diagnostic significance.

  Solution: Implement a standardized scoring system. Research indicates that translating PAX2 expression findings into three categories of loss (complete, partial, minimal to none) provides more clinically relevant information than raw percentage scores .

• Internal controls: Ensuring proper assessment of staining validity.

  Solution: Always evaluate adjacent normal tissue when available, as it provides an internal positive control. The observation that adjacent normal endometrium consistently shows at least partial PAX2 expression can serve as a quality control measure .

How does PAX2 immunohistochemistry compare with other diagnostic markers in urogenital and gynecological pathology?

PAX2 immunohistochemistry offers distinct advantages when integrated into a comprehensive panel with other diagnostic markers:

• Renal cell carcinoma diagnosis:

  • PAX2 has demonstrated high sensitivity for Wilms tumor (97%) and nephrogenic rests (100%)

  • In a complementary panel with other markers like CD10 and RCC marker, PAX2 helps distinguish subtypes of renal cell carcinoma

  • PAX2 is particularly valuable for confirming renal origin in metastatic settings, where morphology alone may be insufficient

• Gynecological pathology:

  • PAX2 loss in endometrial hyperplasia occurs early and shows progressive frequency with increasing lesion severity

  • When combined with PTEN, PAX2 loss can help identify premalignant lesions at high risk for progression

  • The pattern of PAX2 expression in ovarian carcinomas (particularly serous types) can support differential diagnosis

• Complementary marker advantages:

  • PAX2 provides nuclear staining pattern, complementing cytoplasmic or membranous markers

  • The transcription factor nature of PAX2 offers information about cellular differentiation state, complementing proliferation markers

  • Unlike some markers that show gain of expression in neoplasia, PAX2 often shows loss, providing an orthogonal assessment approach

• Differential diagnostic utility:

  • PAX2's consistent expression in normal tissues but variable loss in neoplasia creates a useful contrast

  • The statistically significant difference between lesional tissue and adjacent normal tissue enhances diagnostic confidence

  • Progressive PAX2 loss correlating with histological severity provides supporting evidence for grading decisions

What are the optimal protocols for PAX2 immunohistochemistry?

For optimal PAX2 immunohistochemistry results, consider the following validated protocol recommendations:

For formalin-fixed, paraffin-embedded (FFPE) tissue sections:

• Antibody selection and dilution:

  • For monoclonal antibody EPR8586: Use at 1:250 dilution (5.35 μg/mL)

  • For monoclonal antibody ZR224: Optimal concentrations range from 1:100-200 for concentrate preparations

  • For polyclonal antibodies: Typically use at 1:500-1:2000 for Western blot applications

• Antigen retrieval:

  • Heat-mediated antigen retrieval using Tris-EDTA buffer (pH 9.0) is recommended for optimal results

  • Complete retrieval protocol should include buffer heating followed by cooling period to maximize epitope exposure

• Detection system:

  • For IHC-P: ImmunoHistoProbe one-step HRP Polymer (ready to use) secondary antibody systems have been validated

  • Visualization with DAB (3,3'-diaminobenzidine) provides consistent results for nuclear transcription factors

• Controls:

  • Negative control: Substitute primary antibody with PBS

  • Positive control: Normal kidney tissue sections are ideal for validating PAX2 antibody performance

  • Internal control: Adjacent normal tissue within the same section can serve as reference for staining quality

• Counterstaining:

  • Hematoxylin counterstaining provides optimal nuclear contrast without obscuring PAX2 nuclear positivity

  • Brief counterstaining (30-60 seconds) followed by bluing in running tap water yields best results

How should I troubleshoot PAX2 antibody staining issues?

When encountering problems with PAX2 antibody staining, consider these systematic troubleshooting approaches:

• Weak or absent staining:

  • Verify antibody concentration: PAX2 antibodies may require higher concentrations than typical antibodies; test concentration ranges (e.g., 1:100-1:250 for IHC-P)

  • Enhance antigen retrieval: Extended heat-mediated retrieval using Tris-EDTA buffer (pH 9.0) may improve epitope accessibility

  • Check tissue fixation: Overfixation can mask epitopes; consider extended retrieval times for tissues fixed >24 hours

  • Confirm antibody functionality: Test on validated positive control tissue (kidney) to verify antibody activity

• High background or non-specific staining:

  • Implement blocking steps: Use protein blocking solutions containing BSA (1%) to reduce non-specific binding

  • Adjust antibody concentration: Excessive antibody concentration can cause background; dilute further if background persists

  • Increase wash steps: Additional and longer washes with TBST may reduce non-specific binding

  • Consider alternative antibody: Switch from polyclonal to monoclonal antibody if background persists

• Tissue-specific issues:

  • Endogenous peroxidase activity: Ensure complete quenching with hydrogen peroxide prior to primary antibody application

  • Tissue autofluorescence: For fluorescence-based detection, consider autofluorescence quenching reagents

  • Necrotic tissue areas: Exclude necrotic regions from analysis as they may show non-specific staining

• Inconsistent results between experiments:

  • Standardize fixation times: Maintain consistent fixation protocols (ideally 24 hours in 10% neutral buffered formalin)

  • Control for tissue processing variables: Use the same processing protocol for all comparative samples

  • Use automated platforms: Automated staining systems can reduce inter-run variability

How can I quantify and analyze PAX2 expression patterns?

Quantification and analysis of PAX2 expression patterns require systematic approaches to generate reproducible, meaningful data:

• Categorical scoring systems:

  • Three-tier system: Complete loss (0% cells staining), Partial loss (1-75% cells staining), Minimal to no loss (76-100% cells staining)

  • This approach has demonstrated clinical relevance in studies of endometrial hyperplasia and carcinoma

• Detailed quantitative assessment:

  • Five-tier range system: 0%, 1-25%, 26-50%, 51-75%, or 76-100% positive nuclear staining

  • This granular approach provides more detailed data for research questions requiring precise quantification

• Digital image analysis approaches:

  • Whole slide imaging followed by algorithm-based quantification can reduce observer bias

  • Color deconvolution algorithms can separate DAB (PAX2) from hematoxylin counterstain

  • Nuclear detection algorithms can calculate percentage of positive nuclei with greater precision

• Comparative analysis strategies:

  • Paired analysis: Compare lesional tissue with adjacent normal tissue in the same section

  • This approach controls for technical variables and provides internal reference

  • Statistical significance testing (Fisher's exact test) can validate differences between normal and lesional tissue

• Intensity considerations:

  • Incorporate staining intensity (weak, moderate, strong) alongside percentage of positive cells

  • H-score method: Multiply percentage of cells by intensity score (1, 2, or 3) for values ranging from 0-300

  • This approach captures both extent and intensity of nuclear PAX2 expression

How can PAX2 expression patterns inform research on cancer progression?

PAX2 expression patterns offer valuable insights into carcinogenesis and progression mechanisms:

• Early detection of premalignant changes:

  • PAX2 loss occurs early in endometrial hyperplasia progression

  • This early loss makes PAX2 a potential biomarker for identifying premalignant lesions before morphological changes become pronounced

  • The progressive increase in frequency and completeness of PAX2 loss with increasing histological severity provides a molecular correlate to morphological progression

• Understanding molecular pathogenesis:

  • PAX2 is a transcription factor that regulates developmental processes and cell differentiation

  • Loss of PAX2 suggests disruption of normal differentiation pathways

  • This disruption may represent a key step in carcinogenesis, particularly in tissues where PAX2 normally maintains differentiated phenotypes

• Field effect phenomena:

  • Studies showing partial PAX2 loss in histologically normal tissue adjacent to lesions (16.7% of cases) suggest molecular alterations preceding morphological changes

  • This indicates a "field effect" where molecular alterations extend beyond visibly abnormal tissue

  • Understanding this phenomenon may inform surgical margin assessment and recurrence risk

• Diagnostic and prognostic applications:

  • The statistically significant difference in PAX2 expression between lesional tissue and adjacent normal tissue across diagnostic categories supports its utility as a diagnostic marker

  • The pattern of increasing PAX2 loss with increasing severity suggests potential prognostic value

  • Integration of PAX2 assessment into pathology workflows may enhance diagnostic accuracy and risk stratification

What are the limitations of PAX2 antibodies in research applications?

Despite their utility, PAX2 antibodies have several limitations researchers should consider:

• Isoform specificity limitations:

  • PAX2 has multiple isoforms due to alternative splicing

  • Most commercial antibodies may not distinguish between these isoforms

  • This limitation can affect interpretation when specific isoforms have distinct biological functions

• Cross-reactivity considerations:

  • PAX proteins share conserved domains, particularly in the paired box region

  • Some antibodies may cross-react with related PAX family proteins (PAX5, PAX8)

  • Validation with specific controls is essential to confirm specificity

• Technical variability sources:

  • Fixation time significantly impacts PAX2 immunoreactivity

  • Different antigen retrieval methods yield variable results

  • Antibodies from different manufacturers or clones may produce inconsistent staining patterns

• Interpretation challenges:

  • Distinguishing partial from complete loss requires standardized assessment protocols

  • Heterogeneous staining within samples necessitates evaluation of multiple fields

  • Low-level expression may be difficult to distinguish from background

• Functional assessment limitations:

  • Immunohistochemistry detects protein presence but not functional activity

  • Post-translational modifications affecting PAX2 function are not assessed

  • Complementary approaches (e.g., reporter assays) are needed to assess transcriptional activity

How do different fixation and processing methods affect PAX2 antibody performance?

Fixation and processing significantly impact PAX2 antibody performance, with important considerations for research design:

• Fixative effects:

  • Formalin fixation: 10% neutral buffered formalin is the standard fixative for PAX2 IHC

  • Over-fixation (>48 hours) can mask epitopes and reduce staining intensity

  • Under-fixation (<6 hours) may result in poor tissue morphology and inconsistent staining

  • Non-formalin fixatives (e.g., Bouin's) may require alternative retrieval methods

• Antigen retrieval optimization:

  • Heat-induced epitope retrieval (HIER) with alkaline Tris-EDTA buffer (pH 9.0) provides optimal results for most PAX2 antibodies

  • Acidic citrate buffer (pH 6.0) typically yields weaker nuclear staining

  • Pressure cooker methods generally outperform water bath or microwave approaches

  • Extended retrieval times (20-30 minutes) may be necessary for over-fixed tissues

• Processing variables:

  • Prolonged ethanol dehydration can reduce antigenicity

  • Extended paraffin embedding at high temperatures (>60°C) may damage epitopes

  • Thick sections (>4μm) may show inconsistent staining due to incomplete deparaffinization

  • Storage of cut sections for extended periods (>3 months) before staining reduces antigenicity

• Sample type considerations:

  • FFPE tissues: Most validated protocols are optimized for formalin-fixed, paraffin-embedded tissues

  • Frozen sections: Typically require different antibody concentrations and may show higher background

  • Cell cultures: May require different permeabilization approaches compared to tissue sections

  • Tissue microarrays: Small core size may not represent heterogeneous expression patterns