PXG Antibody

What is PAX2 and why is it important in developmental biology research?

PAX2 is a transcription factor that plays a critical role in stem cell survival and lineage commitment during embryonic development. It functions as a key regulator in the formation of several organ systems, particularly in kidney development and the central nervous system. In research contexts, PAX2 is frequently studied as a marker for developmental processes, stem cell differentiation, and certain pathological conditions . PAX2 is also expressed in various carcinomas where it appears to mediate anti-apoptotic processes, making it relevant for cancer research as well . For developmental biologists, PAX2 antibodies are essential tools for tracking the expression of this protein during critical developmental windows and for identifying specific cell populations during organogenesis.

What sample types can be effectively analyzed using PAX2 antibody?

PAX2 antibody has been validated for multiple sample types in research applications:

Research data demonstrates successful application of PAX2 antibody in paraffin-embedded kidney sections, embryonic stem cells differentiated into early otic lineage, and spinal cord tissues . When working with novel sample types, researchers should conduct preliminary validation experiments with appropriate positive controls.

What species cross-reactivity has been confirmed for PAX2 antibody?

While the product is labeled as Human PAX2 Antibody, experimental validation has confirmed cross-reactivity with multiple species. The antibody has been successfully used to detect PAX2 in human, mouse, and rat samples . Specifically, Western blot experiments have validated reactivity with mouse fetal kidney lysates, immunohistochemistry has confirmed detection in human kidney sections, and immunofluorescence studies have demonstrated effective labeling in rat spinal dorsal horn tissue . This broad cross-species reactivity makes the antibody particularly valuable for comparative studies across different model organisms.

What are the optimal protocols for PAX2 immunohistochemistry in paraffin-embedded tissues?

For optimal PAX2 detection in paraffin-embedded tissues, the following protocol has been validated:

• Tissue preparation: Process tissues using standard fixation (10% neutral buffered formalin) and paraffin embedding

• Sectioning: Cut sections at 5-7 μm thickness

• Antigen retrieval: Perform heat-induced epitope retrieval using Antigen Retrieval Reagent-Basic (pH 8.0-9.0)

• Primary antibody: Apply PAX2 Antibody at 1 μg/mL concentration

• Incubation: Incubate for 1 hour at room temperature

• Detection system: Use Anti-Goat IgG VisUCyte HRP Polymer Antibody

• Visualization: Develop with DAB (brown) and counterstain with hematoxylin (blue)

This protocol has been specifically validated for human kidney samples, where PAX2 shows nuclear localization in convoluted tubules . Researchers should note that optimization may be required for different tissue types, particularly those with lower PAX2 expression levels.

How can PAX2 antibody be used to identify specific neuronal populations in the spinal cord?

PAX2 antibody has been effectively used to identify specific inhibitory interneuron populations in the spinal dorsal horn. Research protocols have demonstrated:

• Co-localization studies: PAX2 (detected with AF3364 antibody) can be used alongside other markers like prodynorphin (PDYN) to characterize specific neuronal subtypes

• Quantification methodology: For accurate quantification, PAX2+ neurons can be counted in defined anatomical regions (e.g., superficial laminae) on both ipsilateral and contralateral sides

• Statistical analysis: Unpaired t-tests have been used to compare PAX2+ neuron numbers between experimental conditions (e.g., after DTX vs. PBS injection)

In published studies, PAX2 antibody has successfully identified a subset of inhibitory neurons when combined with AAV-PdynP-tdT labeling, with approximately 70% of labeled neurons co-expressing PAX2 . This approach allows researchers to target specific neuronal populations for functional studies in pain and sensory processing research.

What are the key considerations for using PAX2 antibody in multiplex immunofluorescence applications?

When using PAX2 antibody in multiplex immunofluorescence studies, researchers should consider:

• Antibody compatibility: Carefully select primary antibodies raised in different host species to avoid cross-reactivity

• Sequential staining protocol:

  • PAX2 detection: Use Goat Anti-Human PAX2 at 10 μg/mL for 3 hours at room temperature

  • Detection with species-specific secondary antibody (e.g., NorthernLights 557-conjugated Anti-Goat IgG)

  • Follow with additional primary antibodies (e.g., VGLUT2, PDYN) raised in different species

• Controls: Include single-stained controls to confirm specificity and rule out cross-reactivity

• Image acquisition: Use sequential scanning to minimize spectral overlap

Published studies have successfully combined PAX2 immunolabeling with markers such as VGLUT2, PDYN, and tdTomato reporter expression in neuronal populations . Quantification of co-expression can be performed by counting double-positive cells as a percentage of total marker-positive cells.

How can PAX2 antibody be utilized in stem cell research and differentiation studies?

PAX2 antibody serves as a valuable tool in stem cell research for:

• Lineage tracing: Tracking cells committed to specific developmental pathways

• Differentiation confirmation: Validating successful differentiation of stem cells into particular cell types

• Temporal expression analysis: Monitoring PAX2 expression changes during differentiation processes

Experimental data shows that PAX2 antibody has been successfully used to detect PAX2 in BG01V human embryonic stem cells differentiated into early otic lineage . For such applications, immunofluorescence protocol optimization typically involves:

• Fixation with 4% paraformaldehyde for 15-20 minutes

• Permeabilization with 0.1-0.3% Triton X-100

• Blocking with 5-10% normal serum

• Primary antibody incubation at 10 μg/mL for 3 hours at room temperature

• Detection with fluorophore-conjugated secondary antibodies

• DAPI counterstaining for nuclear visualization

Researchers should monitor nuclear localization of PAX2 staining as a critical quality control parameter.

What are the common technical challenges when using PAX2 antibody in Western blot applications?

When utilizing PAX2 antibody for Western blot analysis, researchers should address these common challenges:

• Optimizing protein extraction: Nuclear proteins like PAX2 require effective nuclear extraction methods

• Ensuring appropriate sample preparation:

  • Use RIPA or NP-40 based lysis buffers with protease inhibitors

  • Include DNase treatment to reduce sample viscosity

  • Heat samples at 95°C for 5 minutes in reducing buffer

• Gel selection and transfer conditions:

  • Use 10-12% SDS-PAGE gels for optimal separation

  • Transfer to PVDF membrane at 100V for 60-90 minutes

• Detection parameters:

  • Primary antibody concentration: 1 μg/mL

  • Secondary antibody: HRP-conjugated Anti-Goat IgG

  • Visualization: Enhanced chemiluminescence

Western blot analysis with PAX2 antibody typically reveals a specific band at approximately 47 kDa, as demonstrated in mouse fetal kidney lysate samples . Non-specific bands may appear if washing steps are inadequate or if antibody concentration is too high.

How can PAX2 expression data be quantified and statistically analyzed in immunohistochemistry studies?

For rigorous quantification of PAX2 expression in immunohistochemistry studies, researchers should follow these methodological approaches:

• Sampling strategy:

  • Examine multiple non-overlapping fields per section

  • Analyze multiple sections per sample

  • Include biological replicates (n ≥ 3-6 animals/patients)

• Quantification methods:

  • Count total PAX2+ cells in defined anatomical regions

  • Measure staining intensity using calibrated image analysis software

  • Calculate percentage of PAX2+ cells relative to total cell count

• Statistical analysis:

  • For comparison between two groups: Unpaired t-test (if normally distributed)

  • For multiple group comparisons: ANOVA with appropriate post-hoc tests

  • Report data as mean ± SEM with p-values

Published studies have successfully quantified PAX2+ neurons in the superficial laminae of rat spinal dorsal horn, demonstrating significant differences between ipsilateral and contralateral sides following experimental manipulation (p < 0.0001, Unpaired t-test) . This approach provides robust quantitative data for comparative studies.

How is PAX2 antibody used in neurological research, particularly in pain and sensory processing studies?

PAX2 antibody has proven valuable in neurological research focusing on inhibitory interneurons in pain circuits:

• Identification of neuronal subpopulations: PAX2 serves as a marker for inhibitory interneurons in the spinal dorsal horn

• Functional characterization:

  • Colocalization studies with PAX2 and other markers (e.g., PDYN, VGLUT2) help define functional neuronal subsets

  • Approximately 70% of prodynorphin promoter-expressing neurons co-express PAX2, confirming their inhibitory nature

• Manipulation studies:

  • PAX2+ neurons can be selectively targeted using viral vectors and genetic approaches

  • Functional outcomes can be assessed through behavioral testing of sensory modalities

Research has demonstrated that ablation of specific PAX2+ neuronal populations in the spinal dorsal horn affects sensory processing, providing insights into the neural circuits involved in pain perception and mechanical allodynia .

What controls should be included when using PAX2 antibody for experimental validation?

To ensure experimental rigor when using PAX2 antibody, researchers should include these essential controls:

Additionally, when conducting co-expression studies, single-label controls should be included to assess bleed-through in multichannel imaging . These comprehensive controls are essential for publication-quality research and ensure accurate interpretation of PAX2 expression patterns.

How can PAX2 antibody be used in cancer research applications?

PAX2 expression has been identified in various carcinomas, making PAX2 antibody a valuable tool in cancer research:

• Expression profiling:

  • Characterize PAX2 expression patterns in different tumor types

  • Correlate expression with clinical parameters and outcomes

• Mechanistic studies:

  • Investigate PAX2's role in anti-apoptotic mechanisms

  • Examine PAX2's influence on cancer cell proliferation and survival

• Diagnostic applications:

  • Evaluate PAX2 as a potential diagnostic marker

  • Determine if PAX2 expression can distinguish tumor subtypes

• Methodological approach:

  • Tissue microarray analysis of patient samples

  • Immunohistochemical optimization using established protocols

  • Scoring systems based on staining intensity and percentage of positive cells

For cancer studies, researchers should consider combining PAX2 immunostaining with other diagnostic markers to improve specificity and investigate potential correlations with known prognostic indicators.

What are the most effective strategies for troubleshooting weak or absent PAX2 staining?

When encountering weak or absent PAX2 staining, researchers should systematically evaluate:

• Antigen retrieval optimization:

  • Test different antigen retrieval buffers (citrate pH 6.0 vs. EDTA pH 9.0)

  • Extend retrieval time (10-30 minutes)

  • Ensure complete deparaffinization

• Antibody conditions:

  • Increase primary antibody concentration (up to 10 μg/mL)

  • Extend incubation time (overnight at 4°C)

  • Test alternative secondary detection systems

• Tissue quality assessment:

  • Evaluate fixation conditions (overfixation can mask epitopes)

  • Confirm tissue viability with control antibodies

  • Check time between tissue collection and fixation

• Signal amplification:

  • Consider tyramide signal amplification systems

  • Utilize polymer-based detection methods as demonstrated in published protocols

Published protocols have successfully detected PAX2 in human kidney using 1 μg/mL antibody concentration with heat-induced epitope retrieval and HRP polymer detection system . These parameters can serve as a starting point for optimization with other tissue types.