ISL1 Antibody

What cellular structures does ISL1 antibody typically label?

ISL1 antibody primarily labels nuclear structures as ISL1 is a transcription factor with nuclear localization. In immunofluorescence studies, ISL1 shows specific staining localized to the nucleus, as demonstrated in iPS2 human induced pluripotent stem cells differentiated to endocrine progenitor cells . When performing immunostaining experiments, researchers should expect nuclear staining patterns and can use nuclear counterstains like DAPI to confirm proper localization. This nuclear localization is consistent with ISL1's function as a transcription factor that regulates gene expression.

Which tissues are most suitable for ISL1 antibody applications?

Based on expression patterns, ISL1 antibodies work exceptionally well in:

• Embryonic neural tissues, particularly developing motor neurons and cranial ganglia

• Pancreatic islet cells

• Subsets of neurons in the adrenal medulla and dorsal root ganglion

• Retinal tissues (inner nuclear and ganglion cell layers)

• Specific brain regions and the pineal gland

For developmental studies, E9.5-E11.5 mouse embryos have shown excellent results with ISL1 antibody staining at concentrations of approximately 10μg/mL .

What are the recommended protocols for ISL1 antibody in immunofluorescence applications?

For optimal immunofluorescence results with ISL1 antibody:

• Fix cells or tissue sections with 4% paraformaldehyde

• Permeabilize with 0.25% Triton X-100

• Block with appropriate blocking buffer

• Incubate with primary ISL1 antibody:

  • For cell cultures: 10 μg/mL for 3 hours at room temperature

  • For tissue sections: 10 μg/mL (concentration validated on E9.5 and E10.5 mouse embryo sections)

• Wash thoroughly with PBS

• Incubate with appropriate fluorophore-conjugated secondary antibody (e.g., NorthernLights 557-conjugated Anti-Goat IgG for R&D Systems' antibody)

• Counterstain nuclei with DAPI

• Mount and image

This protocol has been successfully applied to both cultured cells and tissue sections, with specific nuclear staining of ISL1 protein.

What are the optimal dilutions for ISL1 antibody in different applications?

Based on validated protocols, the following dilutions are recommended as starting points:

These dilutions provide a starting point, but researchers should optimize conditions for their specific experimental setup and antibody source.

How should ISL1 antibodies be stored to maintain optimal activity?

For optimal preservation of antibody activity:

• Long-term storage: -20°C for up to one year

• Short-term storage/frequent use: 4°C for up to one month

• Avoid repeated freeze-thaw cycles as they can degrade antibody quality

• Most commercial ISL1 antibodies are supplied in buffer containing stabilizers (e.g., 50% glycerol, 0.02% sodium azide, PBS, pH 7.2)

Proper aliquoting upon first thaw can prevent degradation from multiple freeze-thaw cycles and extend the useful life of the antibody.

What are potential causes of weak or absent ISL1 signal in immunostaining?

When troubleshooting weak or absent ISL1 staining:

• Verify expression timing: ISL1 expression is developmentally regulated, so ensure the developmental stage is appropriate (e.g., E9.5-E11.5 for mouse embryos)

• Check fixation protocol: Overfixation may mask epitopes; consider optimization or antigen retrieval

• Antibody concentration: May need optimization; successful staining has been reported at 10 μg/mL for both cultured cells and tissue sections

• Tissue-specific considerations: ISL1 expression varies significantly between tissues and developmental stages; refer to expression patterns in the literature

• Antibody specificity: Ensure the antibody is appropriate for your species; validated antibodies show reactivity with human and mouse ISL1

For embryonic tissues specifically, note that ISL1 expression patterns change significantly during development, with expression in motor neurons and cranial ganglia showing temporal and spatial specificity .

How can I validate the specificity of my ISL1 antibody results?

To confirm specificity of ISL1 antibody staining:

• Positive controls: Use tissues/cells known to express ISL1 (e.g., embryonic motor neurons, pancreatic islet cells)

• Negative controls: Omit primary antibody or use tissues where ISL1 is not expressed

• Knockdown validation: Compare staining in ISL1 knockdown vs. control cells

• Multiple detection methods: Confirm results using different techniques (e.g., IF, WB, IHC)

• Mutant models: Where available, use tissues from Isl1 mutant models as controls, such as the Isl1MCM/Del mutant mouse model which shows significantly reduced ISL1 expression

The search results specifically mention using embryos at E11.5 as a positive control for Western blot detection of ISL1 .

What interfering factors might affect ISL1 antibody binding?

Several factors may interfere with ISL1 antibody binding:

• Post-translational modifications: ISL1 function can be regulated by modifications that might affect epitope accessibility

• Protein-protein interactions: ISL1 functions in complexes with other transcription factors that may mask binding sites

• Fixation artifacts: Certain fixatives may alter epitope structure or accessibility

• Expression levels: Low natural expression may require signal amplification techniques

• Specificity issues: Some antibodies may cross-react with related LIM-homeodomain family members

For optimal results, sample preparation should be carefully controlled and consistent across experiments.

How can ISL1 antibodies be used to study motor neuron development?

ISL1 antibodies provide valuable tools for investigating motor neuron development:

• Lineage tracing: Track motor neuron specification and differentiation by co-staining with other neural markers

• Column identification: Study motor column formation and organization during development

• Mutant analysis: Analyze phenotypes in Isl1 mutant or hypomorphic models

• Axonal projection studies: Combine with neurofilament staining to examine how ISL1 levels affect motor axon trajectories

• Cell fate conversion studies: Investigate the transition of prospective motor neurons to V2 interneurons in Isl1 compound mutants

Research has demonstrated that reduced Isl1 expression in compound mutants leads to improper motor column formation and disrupted axonal trajectories to target muscles, including the diaphragm and axial muscles .

What is the role of ISL1 in pancreatic islet cells and how can antibodies help study this?

ISL1 antibodies are instrumental in researching its role in pancreatic biology:

• Proliferation studies: Track ISL1's promotion of β-cell proliferation through detection of ISL1 overexpression or knockdown

• Transcriptional regulation: Investigate ISL1's binding to c-Myc and CyclinD1 promoters through chromatin immunoprecipitation (ChIP) assays using ISL1 antibodies

• Diabetes models: Study ISL1 expression changes in type 1 and type 2 diabetes mouse models

• Cell cycle analysis: Examine how ISL1 affects cell cycle progression in pancreatic islet cells

• Protein-protein interactions: Use co-immunoprecipitation with ISL1 antibodies to identify interaction partners

Research has shown that ISL1 promotes pancreatic islet cell proliferation by directly binding to and activating c-Myc and CyclinD1 promoters, which can be detected using ChIP assays with ISL1 antibodies .

How can ISL1 antibodies be applied in stem cell research?

ISL1 antibodies offer valuable applications in stem cell biology:

• Differentiation monitoring: Track motor neuron or pancreatic endocrine cell differentiation from stem cells

• Lineage verification: Confirm proper differentiation of pluripotent stem cells into specific lineages

• Sorting applications: Use for FACS to isolate ISL1-positive cell populations

• Functional studies: Assess the impact of ISL1 expression on differentiation potential and cell fate

• Disease modeling: Study ISL1 expression in patient-derived iPSCs differentiated into disease-relevant cell types

The search results specifically mention the detection of ISL1 in human induced pluripotent stem cells differentiated into motor neurons and endocrine progenitor cells using immunofluorescence and Western blot techniques .

What control samples should be included when working with ISL1 antibodies?

For robust experimental design with ISL1 antibodies:

• Positive tissue controls:

  • E9.5-E11.5 mouse embryos (particularly neural tissue)

  • Pancreatic islet cells

  • Human iPSCs differentiated to motor neurons or endocrine progenitors

• Negative controls:

  • Primary antibody omission

  • Non-expressing tissues

  • IgG isotype controls for immunoprecipitation experiments

• Expression manipulation controls:

  • ISL1 overexpression samples

  • ISL1 knockdown samples

  • Isl1 mutant tissues where available

For ChIP experiments specifically, researchers successfully used IgG as a negative control when examining ISL1 binding to c-Myc and CyclinD1 promoters .

How should experimental design differ when studying ISL1 in different tissue contexts?

Tissue-specific considerations for ISL1 antibody experiments:

• Neural tissue:

  • Focus on nuclear staining in specific neuronal populations

  • Consider developmental timing carefully (e.g., E9.5-E11.5 for mouse embryonic studies)

  • Co-staining with neuronal markers can help identify specific subtypes

• Pancreatic tissue:

  • Optimize for detection in islet cells

  • Consider diabetic models for studying expression changes

  • Combine with proliferation markers for functional studies

• Stem cell cultures:

  • Track differentiation stages carefully

  • Compare undifferentiated vs. differentiated states

  • Include appropriate differentiation markers as controls

• Adult tissues:

  • May require different fixation protocols than embryonic tissues

  • Expression levels may be lower than in developmental contexts

For each tissue context, optimization of fixation, permeabilization, and antibody concentration is essential for optimal results.

What considerations are important when designing ChIP experiments with ISL1 antibodies?

For successful ChIP experiments with ISL1 antibodies:

• Antibody selection: Choose ChIP-grade or IP-validated ISL1 antibodies

• Controls: Include IgG controls and input samples

• Target identification: Focus on known ISL1 targets like c-Myc and CyclinD1 promoters

• Cell models: Consider using stable ISL1 overexpressing cell lines to enhance signal

• Quantification method: qPCR analysis of precipitated DNA provides quantitative measurement of binding

• Binding site validation: Confirm binding sites with EMSAs using nuclear extracts

Research has demonstrated successful ChIP experiments showing ISL1 binding to the c-Myc promoter (1.5-fold enrichment compared to IgG) and CyclinD1 promoter (4-fold enrichment) in pancreatic β cells .

How can researchers distinguish between specific and non-specific binding of ISL1 antibodies?

To differentiate specific from non-specific ISL1 antibody binding:

• Pattern analysis: Specific ISL1 staining should be predominantly nuclear

• Competition assays: Preincubation with blocking peptides should reduce specific binding

• Multiple antibodies: Use antibodies from different sources or targeting different epitopes

• Knockout/knockdown validation: Compare with samples where ISL1 expression is reduced

• Binding site mutants: For promoter binding studies, mutation of ISL1 binding sites should prevent complex formation

In EMSA experiments, researchers confirmed specific binding by showing that unlabeled oligonucleotides competed for binding while mutated binding sites failed to compete, and that anti-ISL1 antibody (but not control IgG) disrupted the protein-DNA complex formation .

What factors should be considered when interpreting ISL1 expression levels across different developmental stages?

When analyzing ISL1 expression across development:

• Temporal dynamics: ISL1 expression changes significantly throughout development

• Spatial patterns: Expression is highly tissue-specific and region-specific

• Dosage effects: Graded reduction in ISL1 expression produces different phenotypes

• Technical variables: Standardize fixation, antibody concentration, and imaging parameters

• Comparative analysis: Use consistent methods when comparing different developmental timepoints

Research demonstrates that ISL1 functions in a dose-dependent manner for proper motor neuron specification, maintenance, and axonal projection, with different levels of expression yielding distinct developmental outcomes .

How should researchers address contradictory results between different ISL1 antibody detection methods?

When faced with contradictory results across different methods:

• Method-specific limitations:

  • Western blot detects denatured protein (may miss conformational epitopes)

  • Immunostaining depends on epitope accessibility after fixation

  • ChIP assays are affected by crosslinking efficiency

• Resolution strategies:

  • Validate with multiple antibodies targeting different epitopes

  • Compare polyclonal vs. monoclonal antibodies

  • Implement complementary techniques (e.g., mRNA detection, reporter assays)

  • Validate functionally through overexpression or knockdown

  • Consider post-translational modifications that might affect detection

• Standardization:

  • Use consistent sample preparation across methods

  • Include the same positive and negative controls

  • Validate antibody specificity using knockout/knockdown samples