CHX10 Antibody

What is CHX10 protein and why is it important in research?

CHX10, also known as Visual System Homeobox 2 (VSX2), is a transcription factor with a canonical amino acid length of 361 residues and a protein mass of 39.4 kilodaltons in humans. It belongs to the Paired homeobox protein family and is predominantly localized in the nucleus of cells, with notable expression in the retina . CHX10 plays a crucial role in mammalian eye development, particularly influencing retinal progenitor cell proliferation and bipolar cell specification . Its importance in research stems from its essential function in early retinal development, making it a vital target for studies related to retinal biology and associated disorders .

What are the known functions of CHX10 in retinal development?

CHX10 functions primarily in the regulation of transcription during retinal development . It shows high expression in uncommitted retinal progenitor cells, indicating its significance in early developmental stages . CHX10 expression is initially localized in early retinal neuroepithelium and later becomes restricted to bipolar cells, maintaining low levels in mature retina . The protein is particularly important for neuroretina formation and inner nuclear layer development and maintenance . Research has demonstrated that CHX10 interacts with other key factors in the retinal development pathway, highlighting its role in establishing proper retinal architecture and function .

What detection methods are compatible with CHX10 antibodies?

CHX10 antibodies are versatile tools compatible with multiple detection methods. Based on commercially available antibodies, researchers can utilize these antibodies for western blotting (WB), immunoprecipitation (IP), immunofluorescence (IF), immunohistochemistry (IHC), and enzyme-linked immunosorbent assay (ELISA) . The choice of detection method should be guided by the specific research question, with western blotting being particularly useful for protein expression quantification and immunofluorescence for localization studies within retinal tissue sections.

How should researchers optimize Western blotting protocols for CHX10 detection?

For optimal Western blotting of CHX10, researchers should consider the following protocol modifications:

• Sample preparation: Nuclear protein extraction techniques are recommended given CHX10's nuclear localization

• Gel percentage: Use 10-12% polyacrylamide gels suitable for resolving the ~39.4 kDa CHX10 protein

• Transfer conditions: Optimize transfer time and voltage for complete transfer of nuclear proteins

• Blocking conditions: Use 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature

• Primary antibody incubation: Dilute antibody according to manufacturer recommendations (typically 1:500-1:1000) and incubate overnight at 4°C

• Secondary antibody selection: Choose a species-appropriate HRP-conjugated secondary antibody for detection

• Signal development: Consider enhanced chemiluminescence detection for sensitivity

Researchers should validate their protocol with positive controls such as retinal tissue lysates where CHX10 expression is well-characterized.

What are the optimal conditions for immunofluorescence using CHX10 antibodies?

For immunofluorescence applications with CHX10 antibodies, consider these optimization strategies:

• Fixation: 4% paraformaldehyde for 15-20 minutes preserves CHX10 epitopes while maintaining tissue architecture

• Permeabilization: 0.2-0.3% Triton X-100 for 10 minutes to allow antibody access to nuclear CHX10

• Blocking: 5-10% normal serum (from the species of secondary antibody) with 1% BSA for 1 hour

• Primary antibody: Dilute according to manufacturer recommendations (typically 1:100-1:500) and incubate overnight at 4°C

• Secondary antibody: Use fluorophore-conjugated antibodies (Alexa Fluor series or FITC) at 1:500-1:1000 dilution

• Counterstaining: DAPI nuclear stain helps confirm nuclear localization of CHX10

• Mounting: Use anti-fade mounting medium to preserve fluorescence

Bipolar cells in retinal sections and retinal progenitor cells in developing tissue serve as positive controls.

What species compatibility exists for commercial CHX10 antibodies?

Most commercially available CHX10 antibodies demonstrate cross-reactivity with human, mouse, and rat species . This cross-reactivity reflects the high conservation of the CHX10/VSX2 protein sequence across mammalian species. When designing experiments involving other species, researchers should verify antibody compatibility through literature review or preliminary validation studies. Antibody datasheets typically list confirmed species reactivity, though untested species may still show compatibility due to sequence homology.

How do expression patterns of CHX10 differ across development and species?

CHX10 expression patterns show both temporal and species-specific variations:

• Developmental differences:

  • High expression in uncommitted retinal progenitor cells during early development

  • Restricted expression in bipolar cells in mature retina

  • Maintained at lower levels in mature retina compared to developmental stages

• Species considerations:

  • Similar expression patterns in retinal bipolar cells across mammals

  • Timing of expression may vary between faster-developing (rodent) and slower-developing (human) retinas

  • Expression intensity may differ between species due to evolutionary adaptations

These differences necessitate careful consideration when designing comparative studies between species or developmental timepoints.

How can CHX10 antibodies be utilized for developmental studies?

CHX10 antibodies are valuable tools for tracking retinal development through various experimental approaches:

• Temporal expression profiling:

  • Sequential immunohistochemistry at different developmental stages

  • Western blot analysis of CHX10 expression throughout retinal development

  • Flow cytometry to quantify CHX10-positive cells during development

• Lineage tracing:

  • Co-staining with progenitor markers to identify CHX10's role in retinal progenitor specification

  • Sequential labeling to track the fate of CHX10-expressing cells

• Functional studies:

  • Chromatin immunoprecipitation (ChIP) assays to identify CHX10 target genes

  • Combining CHX10 immunostaining with BrdU labeling to assess proliferation

These approaches enable detailed characterization of CHX10's role in retinogenesis and bipolar cell specification.

What are effective approaches for multiplex staining with CHX10 antibodies?

For multiplex staining with CHX10 antibodies, researchers should consider:

• Primary antibody selection:

  • Choose CHX10 antibodies from different host species than other target antibodies

  • Alternatively, use directly conjugated CHX10 antibodies (FITC, PE, or Alexa Fluor conjugates)

• Sequential staining protocol:

  • Perform CHX10 staining first as it requires nuclear localization

  • Block with excess secondary antibody before proceeding to subsequent markers

  • Consider tyramide signal amplification for weak signals

• Recommended marker combinations:

  • CHX10 with bipolar cell markers (PKCα, Islet1) for mature retina studies

  • CHX10 with progenitor markers (Ki67, Nestin) for developmental studies

  • CHX10 with other transcription factors (Pax6, Otx2) for regulatory network analysis

• Image acquisition:

  • Use spectral imaging to separate overlapping fluorophores

  • Employ sequential scanning in confocal microscopy to prevent bleed-through

How can researchers validate CHX10 antibody specificity?

Rigorous validation ensures reliable experimental outcomes. For CHX10 antibodies, implement these validation approaches:

• Positive controls:

  • Retinal tissue sections with known CHX10 expression patterns

  • Cell lines with confirmed CHX10 expression

• Negative controls:

  • Tissues known to lack CHX10 expression

  • CHX10 knockout/knockdown samples when available

  • Primary antibody omission controls

• Peptide competition assay:

  • Pre-incubate antibody with immunizing peptide

  • Observe elimination of specific signal

• Multiple antibody validation:

  • Compare staining patterns using antibodies targeting different CHX10 epitopes

  • Cross-validate with mRNA expression data (in situ hybridization or RNA-seq)

Properly documented validation enhances reproducibility and confidence in experimental findings.

What are common issues in CHX10 immunostaining and how can they be resolved?

How should researchers address contradictory data when using different CHX10 antibody clones?

When facing contradictory results using different CHX10 antibody clones (such as D-11 and E-12) , researchers should systematically investigate the source of discrepancy:

• Epitope mapping:

  • Determine if antibodies recognize different domains of CHX10

  • Consider whether post-translational modifications might affect epitope accessibility

• Validation with multiple approaches:

  • Corroborate protein expression with mRNA analysis

  • Employ knockout/knockdown controls when possible

  • Use recombinant CHX10 protein as a standard

• Experimental conditions assessment:

  • Evaluate whether differences emerge under specific fixation conditions

  • Test both antibodies under identical protocols

  • Consider species-specific differences in epitope conservation

• Reporting practices:

  • Document all validation steps thoroughly

  • Report the specific antibody clone, catalog number, and lot in publications

  • Acknowledge potential limitations in data interpretation

How can CHX10 antibodies contribute to retinal disease research?

CHX10 antibodies offer valuable insights into retinal pathologies through several research applications:

• Developmental disorders:

  • Characterizing CHX10 expression in microphthalmia and other congenital visual impairments

  • Studying CHX10 mutations and their impact on retinal progenitor proliferation

• Degenerative conditions:

  • Tracking bipolar cell loss in retinal degenerative diseases

  • Assessing CHX10 expression changes in response to retinal stress

• Regenerative medicine:

  • Monitoring CHX10 expression during differentiation of stem cells into retinal lineages

  • Evaluating bipolar cell generation in retinal organoids

• Biomarker development:

  • Using CHX10 as a marker for specific retinal cell populations in disease progression

  • Correlating CHX10 expression patterns with visual function outcomes

The precise regulation of CHX10 expression is vital for proper retinal architecture and function, making antibodies against this protein essential tools for understanding both normal development and pathological conditions .

What innovative methods are being developed for CHX10 detection and analysis?

Emerging methods for CHX10 detection extend beyond traditional antibody applications:

• Single-cell analysis:

  • Single-cell RNA sequencing paired with protein analysis for correlation studies

  • Mass cytometry (CyTOF) for simultaneous detection of CHX10 with dozens of other markers

• Advanced imaging:

  • Super-resolution microscopy for precise subcellular localization

  • Light-sheet microscopy for whole-tissue CHX10 mapping

  • Live cell imaging using knock-in fluorescent reporters

• Proximity-based methods:

  • Proximity ligation assays to detect CHX10 protein interactions

  • Chromatin immunoprecipitation sequencing (ChIP-seq) for genome-wide binding site identification

• Functional genomics integration:

  • CRISPR screens combined with CHX10 immunophenotyping

  • Multiomics approaches correlating CHX10 protein levels with transcriptomic and epigenomic data

These innovative approaches will enhance our understanding of CHX10's multifaceted roles in retinal biology and development.