GLIS3 Antibody, FITC conjugated

What is GLIS3 and why is it important in research?

GLIS3 is a Krüppel-like zinc finger transcription factor that functions as both a repressor and activator of transcription. It binds to the consensus sequence 5'-GACCACCCAC-3' to regulate gene expression . GLIS3 plays critical roles in pancreatic development and beta-cell function, with mutations in the GLIS3 gene linked to neonatal diabetes . Research on GLIS3 is particularly important for understanding pancreatic islet development and pathogenesis of diabetes, as GLIS3 directly transactivates Neurogenin 3 (Neurog3), a key transcription factor in endocrine pancreas lineage determination .

What are the optimal applications for FITC-conjugated GLIS3 antibodies?

FITC-conjugated GLIS3 antibodies are particularly well-suited for immunofluorescence applications including:

• Flow cytometry for quantifying GLIS3-expressing cells

• Immunocytochemistry/immunofluorescence (ICC/IF) on cultured cells

• Fluorescence microscopy of tissue sections

• Live-cell imaging (for certain membrane-associated fractions)

Based on validated applications of unconjugated GLIS3 antibodies, optimal results are typically achieved in ICC/IF applications, such as those demonstrated in A549 cells (human lung carcinoma cell line) where GLIS3 antibodies have been successfully used at concentrations of 2 μg/ml .

What is the typical subcellular localization pattern of GLIS3?

GLIS3 predominantly localizes to the nucleus, consistent with its function as a transcription factor. When visualized using FITC-conjugated antibodies, GLIS3 typically shows a nuclear staining pattern with some nucleolar exclusion. In certain developmental contexts or pathological conditions, cytoplasmic retention may be observed. During ChIP-seq experiments, GLIS3 has been shown to bind within proximal regulatory regions (defined as within 5 kb upstream or downstream of transcription start sites) of target genes . This nuclear localization is critical for its function in transcriptional regulation of genes like Neurog3 and members of the WNT signaling pathway .

How should fixation and permeabilization be optimized for FITC-conjugated GLIS3 antibody staining?

For optimal FITC-conjugated GLIS3 antibody staining, the following fixation and permeabilization protocols are recommended based on validated applications:

For cell cultures (ICC/IF):

• Fixation: 4% paraformaldehyde (PFA) for 15-20 minutes at room temperature

• Permeabilization: 0.1-0.3% Triton X-100 for 10 minutes at room temperature

This approach has been validated in A549 cells, where PFA fixation followed by Triton X-100 permeabilization allowed effective GLIS3 detection .

For tissue sections (IHC):

• Formalin-fixed paraffin-embedded (FFPE) samples are suitable

• Antigen retrieval: Citrate buffer (pH 6.0) heat-induced epitope retrieval

• Typical dilutions for primary antibody: 1/50 (as demonstrated in human thyroid tissue)

For FITC-conjugated antibodies specifically, minimize exposure to light throughout the protocol to prevent photobleaching of the fluorophore.

What controls are essential when using FITC-conjugated GLIS3 antibodies?

When conducting experiments with FITC-conjugated GLIS3 antibodies, the following controls are essential:

• Negative controls:

  • Isotype control (FITC-conjugated IgG from same species as the GLIS3 antibody)

  • Secondary antibody-only control (if using indirect detection)

  • GLIS3 knockout or knockdown samples (ideally GLIS3-/- cells)

• Positive controls:

  • Cell lines with known GLIS3 expression (e.g., pancreatic islet cells, thyroid tissue)

  • Overexpression systems (cells transfected with GLIS3 expression constructs)

• Specificity controls:

  • Peptide competition assay using the immunizing peptide (GLIS3 aa 550-700)

  • Comparison with alternative GLIS3 antibody clones

In particular, GLIS3-/- mouse models have shown >95% reduction in NEUROG3-positive cells compared to wild-type controls, providing a strong validation system for antibody specificity .

How can FITC-conjugated GLIS3 antibodies be used in multiplexed imaging systems?

For multiplexed imaging with FITC-conjugated GLIS3 antibodies, consider the following approach:

• Compatible fluorophore selection:

  • FITC emission (green, ~520 nm) can be combined with:

    • Red fluorophores (e.g., Texas Red, Cy3) for transcription factors that colocalize with GLIS3

    • Far-red fluorophores (e.g., Cy5, Alexa Fluor 647) for additional markers

    • DAPI (blue) for nuclear counterstaining

• Recommended multiplex combinations:

  • GLIS3-FITC + NEUROG3-Cy3 + DAPI: To study the regulatory relationship between GLIS3 and NEUROG3

  • GLIS3-FITC + HNF6-Cy5 + FOXA2-Texas Red + DAPI: To examine the transcription factor complex that synergistically activates Neurog3

• Sequential staining protocol:

  • Fix and permeabilize samples

  • Block with appropriate serum

  • Apply directly conjugated antibodies simultaneously if using different species

  • For same-species antibodies, use sequential staining with blocked fab fragments between steps

Controls for spectral overlap should be included to ensure proper separation of signals.

How can ChIP assays be optimized using FITC-conjugated GLIS3 antibodies?

While FITC conjugation is not typically used for ChIP applications (as it may interfere with antigen binding), the following protocol adaptations can enable chromatin studies with GLIS3 antibodies:

• ChIP-seq optimization:

  • Use unconjugated GLIS3 antibody for immunoprecipitation

  • Perform ChIP using 1-5 μg antibody per 25 μg chromatin

  • Focus on identified GLIS3 response elements (GLIS3REs), particularly the consensus sequence 5'-GACCACCCAC-3'

• Known GLIS3 binding sites:

  • In the Neurog3 promoter, multiple GLIS3REs have been identified at positions -2,862, -2,718, -1,160, and -1,117 relative to the transcription start site

  • The -2,718 site shows highest conservation across species and strongest binding in ChIP assays

• Data analysis parameters:

  • For ChIP-seq data alignment, use parameters similar to those employed in previous studies:

    • Map to reference genome with maximum two mismatches

    • Retain only uniquely mapped reads

    • Normalize to reads per million (RPM)

    • Define binding sites using peak calling algorithms like SISSRs

For visualization of GLIS3 binding post-ChIP, FITC-labeled secondary antibodies can be used in combination with confocal microscopy to correlate binding with nuclear architecture.

What approaches can resolve contradictory results when detecting GLIS3 in different developmental stages?

When encountering contradictory results in GLIS3 detection across developmental stages, employ these systematic troubleshooting approaches:

• Developmental timing considerations:

  • GLIS3 expression is temporally regulated, particularly during pancreatic development

  • In mouse models, significant differences in NEUROG3-positive cells have been observed between E12.5 and E13.5 embryos

  • Use precise developmental staging and time-course experiments

• Epitope masking resolution:

  • Different fixation protocols may mask the GLIS3 epitope

  • Test multiple antigen retrieval methods (heat-induced vs. enzymatic)

  • Consider using antibodies targeting different GLIS3 epitopes

• Protein interaction effects:

  • GLIS3 interacts with multiple transcription factors including HNF6 and FOXA2

  • These interactions may mask antibody binding sites

  • Use protein-protein interaction disruptors or alternative fixation methods

• Cross-validation approaches:

  • Combine antibody detection with mRNA analysis (e.g., in situ hybridization)

  • Use genetic reporter systems (e.g., GLIS3-EGFP knock-in models)

  • Employ multiple antibody clones targeting different GLIS3 epitopes

How can FITC-conjugated GLIS3 antibodies be utilized in stem cell differentiation protocols?

FITC-conjugated GLIS3 antibodies offer valuable tools for monitoring and optimizing stem cell differentiation protocols:

• Differentiation monitoring:

  • GLIS3 has been shown to direct differentiation of human embryonic stem cells (hESCs)

  • Use FITC-conjugated GLIS3 antibodies for:

    • Flow cytometry to quantify GLIS3-expressing populations during differentiation

    • Live-cell sorting of GLIS3-positive progenitors

    • Time-lapse imaging to track GLIS3 expression dynamics

• Pancreatic differentiation optimization:

  • GLIS3 plays a critical role in pancreatic islet differentiation through NEUROG3 activation

  • Design differentiation protocol checkpoints based on GLIS3 expression

  • Monitor co-expression of GLIS3 with other pancreatic transcription factors

• Differentiation efficiency quantification:

  • Use flow cytometry with FITC-conjugated GLIS3 antibodies to determine percentage of cells expressing GLIS3 at each differentiation stage

  • Correlate GLIS3 expression patterns with functional maturation markers

  • Create quantitative benchmarks for successful differentiation based on GLIS3 signal intensity and localization

What are the best approaches for validating GLIS3 antibody specificity in knockout/knockdown systems?

To rigorously validate GLIS3 antibody specificity, especially for FITC-conjugated variants, implement these validation strategies:

• Genetic validation approaches:

  • GLIS3 knockout models: Complete absence of signal should be observed in GLIS3-/- samples

  • GLIS3 knockdown: Signal intensity should correlate with degree of knockdown

  • Overexpression systems: Increased signal should correspond with GLIS3 overexpression

• Molecular validation methods:

  • Western blot: Confirm single band of appropriate molecular weight (~90 kDa)

  • Mass spectrometry: Verify immunoprecipitated protein identity

  • RNA-protein correlation: Compare antibody signal with mRNA expression levels

• Performance metrics to assess:

  • Signal-to-noise ratio in positive vs. negative samples

  • Dynamic range of detection

  • Reproducibility across technical replicates

  • Consistency between antibody lots

Studies in GLIS3-/- mouse models have demonstrated >95% reduction in NEUROG3-positive cells, providing a benchmark for antibody validation in developmental contexts .

How can signal amplification methods enhance detection of low-abundance GLIS3 protein?

For enhancing detection of low-abundance GLIS3 protein, particularly in developmental contexts, consider these signal amplification strategies:

• Enzymatic amplification methods:

  • Tyramide Signal Amplification (TSA): Can enhance FITC signal 10-50 fold

  • Implementation protocol:

    1. Use biotinylated primary or secondary antibody

    2. Add streptavidin-HRP

    3. Apply FITC-tyramide substrate

    4. Allow brief reaction (5-10 minutes)

    5. Stop reaction and proceed with imaging

• Multi-layer detection systems:

  • Primary antibody → Biotinylated secondary → Streptavidin-FITC

  • Each layer increases sensitivity approximately 2-3 fold

• Signal-to-noise optimization:

  • Extended blocking (2-3 hours) with 5% serum + 1% BSA

  • Longer, more dilute primary antibody incubation (overnight at 4°C)

  • Inclusion of 0.1% Triton X-100 in wash buffers

For quantitative applications, generate standard curves using known quantities of recombinant GLIS3 protein to determine detection limits and linear range of amplified signals.

What is the optimal preservation method for maintaining FITC fluorescence during long-term storage of stained samples?

To maintain FITC fluorescence integrity in stained samples over extended periods, implement these preservation strategies:

• Mounting medium selection:

  • Use anti-fade mounting media containing:

    • p-Phenylenediamine (PPD, 0.1-1%)

    • n-Propyl gallate (0.5%)

    • DABCO (1,4-diazabicyclo[2.2.2]octane, 2.5%)

  • Commercial options: ProLong Gold, Vectashield, or FluorSave

• Storage conditions optimization:

  • Temperature: -20°C for long-term storage

  • Humidity: Store with desiccant to prevent moisture

  • Light exposure: Shield from light using opaque containers

  • Seal edges of coverslips with nail polish to prevent oxidation

• Stability enhancement timeline:

  • Short-term (1-7 days): 4°C in dark conditions

  • Medium-term (1-6 months): -20°C with anti-fade mounting media

  • Long-term (6+ months): -80°C after post-fixation with 4% PFA

• Re-staining protocols:

  • For samples requiring re-examination after signal fading:

    1. Remove coverslip in PBS

    2. Wash extensively to remove mounting medium

    3. Re-stain with freshly prepared FITC-conjugated antibody

    4. Mount with fresh anti-fade medium

How can quantitative analysis of GLIS3 expression be correlated with functional outcomes?

To establish meaningful correlations between GLIS3 expression and functional outcomes:

• Image analysis parameters:

  • Nuclear intensity measurements (mean fluorescence intensity)

  • Nuclear-to-cytoplasmic ratio of GLIS3 signal

  • Percentage of GLIS3-positive cells in a population

  • Colocalization coefficients with interacting proteins (e.g., HNF6, FOXA2)

• Functional correlation approaches:

  • Gene expression correlation: Measure expression of GLIS3 target genes (e.g., Neurog3) using qRT-PCR or RNA-seq

  • Reporter assays: Use GLIS3RE-driven reporters containing the consensus sequence (5'-GACCACCCAC-3')

  • Physiological outcomes: For pancreatic studies, correlate with insulin production or glucose responsiveness

• Statistical analysis workflow:

  • Determine normality of data distribution

  • Apply appropriate parametric or non-parametric tests

  • Use regression analysis to establish quantitative relationships

  • Account for confounding variables through multivariate analysis

In developmental studies, correlate GLIS3 expression patterns with known benchmarks such as the 95% reduction in NEUROG3-positive cells observed in GLIS3-/- embryos at E12.5 .

What computational tools are most effective for analyzing GLIS3 binding patterns from ChIP-seq data?

For effective analysis of GLIS3 binding patterns from ChIP-seq data, implement these computational approaches:

• Recommended analysis pipeline:

  • Alignment: Bowtie (version 0.12.8 or newer) with parameters allowing maximum two mismatches and unique genomic mapping

  • Peak calling: SISSRs with default settings

  • Visualization: Normalize to reads per million (RPM) and display as histograms

  • Motif analysis: MEME-ChIP or HOMER to identify enriched DNA binding motifs

• GLIS3 binding pattern characterization:

  • Analyze genomic distribution relative to transcription start sites

  • Define proximal regulatory regions (within 5 kb upstream or downstream of TSSs)

  • Identify distal enhancer regions through histone mark correlation

• Integration with other genomic datasets:

  • RNA-seq to correlate binding with gene expression changes

  • ATAC-seq or DNase-seq to correlate with chromatin accessibility

  • Hi-C data to understand 3D chromatin organization around GLIS3 binding sites

• Visualization tools:

  • UCSC Genome Browser for viewing binding profiles

  • IGV (Integrative Genomics Viewer) for detailed inspection

  • Heatmaps of binding intensity across different conditions

These approaches have successfully identified GLIS3 binding to WNT gene promoters and established GLIS3 as a transcriptional activator of WNT signaling .

How can single-cell analysis be combined with FITC-conjugated GLIS3 antibody staining?

To effectively integrate FITC-conjugated GLIS3 antibody detection with single-cell analysis:

• Single-cell preparation optimization:

  • Gentle dissociation protocols to preserve nuclear integrity

  • Use of nuclear membrane stabilizers during fixation

  • Careful titration of antibody concentration to minimize background

• Flow cytometry applications:

  • Single-cell sorting of GLIS3-FITC positive populations

  • Index sorting to correlate GLIS3 expression with subsequent single-cell RNA-seq

  • Protocol for pancreatic progenitors:

    1. Dissociate tissue with TrypLE (15 min, 37°C)

    2. Fix with 2% PFA (10 min, RT)

    3. Permeabilize with 0.1% Triton X-100

    4. Stain with FITC-conjugated GLIS3 antibody (1:100)

    5. Sort cells based on GLIS3 expression levels

• CyTOF (mass cytometry) integration:

  • Metal-tagged GLIS3 antibodies can provide higher multiplexing capacity

  • Comparison with FITC-conjugated antibodies shows similar detection patterns

  • Allows simultaneous detection of >30 markers

• Single-cell sequencing correlation:

  • GLIS3-positive cells can be indexed and subjected to scRNA-seq

  • GLIS3 protein levels (by FITC intensity) can be correlated with mRNA expression

  • Regulatory network reconstruction by combining protein and RNA data

This integrated approach is particularly valuable for studying heterogeneous populations during pancreatic development and disease progression.

How do FITC-conjugated GLIS3 antibodies compare with alternative detection methods?

A systematic comparison of GLIS3 detection methods reveals distinct advantages and limitations:

FITC-conjugated antibodies offer a balance of specificity and sensitivity, with the advantage of single-step staining protocols. For developmental studies, combining FITC-conjugated GLIS3 antibodies with lineage markers has proven particularly effective for tracking differentiation trajectories.

What are the optimal FITC-conjugated GLIS3 antibody applications in developmental biology research?

FITC-conjugated GLIS3 antibodies offer specific advantages in developmental biology research contexts:

• Embryonic pancreas development studies:

  • Track GLIS3 expression during critical developmental windows (E12.5-E13.5)

  • Monitor co-expression with NEUROG3 to evaluate proper endocrine lineage specification

  • Assess dysregulation in diabetic models

  • Application protocol:

    1. Section embryonic pancreatic tissue (5-8 μm)

    2. Perform heat-mediated antigen retrieval

    3. Block with 10% serum

    4. Apply FITC-conjugated GLIS3 antibody (1:50-1:100)

    5. Counterstain with DAPI and markers of interest

• Stem cell differentiation monitoring:

  • Quantify GLIS3 expression during directed differentiation protocols

  • Isolate GLIS3-positive progenitor populations

  • Validate proper developmental trajectory toward pancreatic lineages

• Organ morphogenesis assessment:

  • Beyond pancreas, GLIS3 plays roles in kidney, liver, and thyroid development

  • FITC-conjugated antibodies allow simultaneous assessment of GLIS3 with tissue-specific markers

These applications have been instrumental in establishing GLIS3's role in pancreatic islet differentiation via direct transactivation of Neurog3, providing insights into neonatal diabetes pathogenesis .

How can FITC-conjugated GLIS3 antibodies be used to resolve conflicting genomic data?

FITC-conjugated GLIS3 antibodies can help resolve conflicting genomic data through these integrated approaches:

• Protein-centric validation of genomic findings:

  • Use FITC-conjugated GLIS3 antibodies to verify:

    • Protein expression corresponds with mRNA levels

    • Protein localization matches predicted function

    • Developmental timing of expression aligns with genomic data

• Multi-omics integration strategies:

  • Combine FITC-GLIS3 immunofluorescence with:

    • Single-cell RNA-seq to correlate protein and mRNA at cellular level

    • ChIP-seq to confirm binding at predicted target sites

    • ATAC-seq to correlate GLIS3 binding with chromatin accessibility

• Specific conflict resolution approaches:

  • For discrepancies in temporal expression:

    • Perform detailed time-course experiments with FITC-GLIS3 antibodies

    • Compare with mRNA dynamics using RT-PCR or RNA-seq

  • For binding site conflicts:

    • Combine FITC-GLIS3 immunofluorescence with DNA FISH to visualize co-localization

    • Perform sequential ChIP followed by immunofluorescence validation

• Case study: GLIS3-Neurog3 regulatory axis:

  • ChIP experiments have identified multiple GLIS3REs in the Neurog3 promoter

  • Using FITC-GLIS3 antibodies to track protein expression in wild-type vs. mutant models provides functional validation of genomic binding data

  • This approach confirmed that GLIS3 directly transactivates Neurog3, resolving previously conflicting models of pancreatic endocrine lineage specification