TBX1 Antibody, FITC conjugated

What is the functional significance of TBX1 in developmental biology?

TBX1 is a critical transcription factor that regulates multiple aspects of embryonic development. Research demonstrates that TBX1 functions as a key regulator of cardiac progenitor cell (CPC) homeostasis by positively modulating their proliferation while negatively regulating their differentiation . In multipotent heart progenitors, TBX1 stimulates proliferation while maintaining cells in an undifferentiated state. TBX1's expression pattern is largely restricted to the second heart field (SHF), where it overlaps with the SHF marker Isl1, but importantly, TBX1 expression disappears upon differentiation . Additionally, TBX1 plays crucial roles in craniofacial development through regulation of microRNA-96-5p and interaction with PITX2 , and regulates brain vascularization by controlling VEGFR3 and DLL4 genes in brain endothelial cells .

How does TBX1 regulate cell differentiation at the molecular level?

TBX1 regulates cell differentiation through multiple molecular mechanisms:

• Protein-protein interactions: TBX1 binds to Serum Response Factor (SRF), a master regulator of muscle differentiation, and negatively regulates its levels . TBX1 also interacts with PITX2 through its N-terminal domain to repress PITX2 transcriptional activity .

• Transcriptional regulation: TBX1 directly binds to regulatory regions of target genes. For example, it binds to the promoter of miR-96 and represses its expression .

• Modulation of signaling pathways: In lymphatic endothelial cells, TBX1 regulates genes like Dtx1, which affects Notch signaling. When TBX1 is depleted, Dtx1 expression decreases and Notch1 expression increases, which inversely correlates with reduced VEGFR3 expression .

The combined effect of these molecular mechanisms maintains cells in a proliferative, undifferentiated state. When TBX1 expression is lost, premature differentiation occurs, as evidenced by expanded expression domains of differentiation markers like α-SMA, cardiac actin, MF20, and cardiac Troponin T (cTnT) in Tbx1-/- embryos .

What cellular compartments typically express TBX1 protein?

TBX1 expression shows specific tissue and developmental stage localization patterns:

• Cardiovascular system: TBX1 is expressed in multipotent heart progenitors in the second heart field (SHF) but not in differentiated myocardial cells . Immunostaining of mouse embryos at different stages (5-22 somites) showed overlap between TBX1 and the SHF marker Isl1, but TBX1 appears more restricted to the undifferentiated SHF cells, especially at 22 somites .

• Craniofacial structures: TBX1 is expressed in dental progenitor cells and contributes to craniofacial development .

• Vascular system: TBX1 is specifically expressed in lymphatic endothelial cells (LECs) and brain endothelial cells where it regulates vascularization .

• Pharyngeal arches: The expression patterns of TBX1 and GATA6 overlap in the pharyngeal arches of human embryos .

Importantly, TBX1 expression typically disappears when cells begin differentiation, making it a valuable marker for identifying undifferentiated progenitor populations.

What are the optimal fixation protocols for TBX1 immunofluorescence with FITC-conjugated antibodies?

For optimal TBX1 immunofluorescence using FITC-conjugated antibodies, the following fixation protocol is recommended based on research methodologies:

• Fixation: For tissue sections, 4% paraformaldehyde in PBS for 15-20 minutes at room temperature has shown optimal results. For cultured cells, 10 minutes in 4% paraformaldehyde is typically sufficient.

• Permeabilization: Use 0.2% Triton X-100 in PBS for 10 minutes at room temperature.

• Blocking: Incubate with 5-10% normal serum (matched to the secondary antibody species if using non-conjugated primary antibodies) with 1% BSA in PBS for 1 hour at room temperature.

• Antibody incubation: For FITC-conjugated TBX1 antibodies, dilute to manufacturer's recommended concentration (typically 1:100 to 1:500) in blocking solution and incubate overnight at 4°C in a humidified chamber protected from light to prevent photobleaching of the FITC fluorophore.

• Washing: Wash 3-5 times with PBS containing 0.1% Tween-20.

• Counterstaining: DAPI (1:1000) for nuclear visualization.

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

When co-staining for other markers, research protocols have successfully combined TBX1 detection with markers such as Isl1, α-SMA, and other differentiation markers .

How can TBX1 antibody be used to identify cardiac progenitor populations?

TBX1 antibody is a valuable tool for identifying undifferentiated cardiac progenitor populations, particularly in the second heart field (SHF). Based on published research protocols:

• Dual immunostaining approach: Co-stain tissue sections with FITC-conjugated TBX1 antibody and markers for cardiac progenitors such as Isl1. Research has shown that while Isl1 is expressed more extensively, TBX1 expression is more restricted to the undifferentiated SHF cells .

• Negative selection strategy: Use TBX1 antibody in combination with differentiation markers (α-SMA, cardiac actin, MF20, cTnT). Studies have demonstrated essentially no overlap between TBX1 and differentiation markers like α-SMA at developmental stages of 16 and 22 somites .

• Flow cytometry application: For isolating cardiac progenitor populations, FITC-conjugated TBX1 antibody can be used in FACS protocols after proper tissue dissociation. Cells expressing TBX1 represent undifferentiated progenitors, as TBX1 expression disappears with the onset of muscle markers.

• Verification of multipotency: Research has shown that TBX1-expressing cells can be verified as multipotent through clonal analysis. In culture, these cells can give rise to progeny expressing endothelial (Pecam1), smooth muscle (SM-MHC), and cardiomyocyte (cTnT) markers .

For accurate identification, TBX1 antibody staining should be combined with RT-PCR verification of cardiac progenitor markers such as Nkx2.5, Isl1, and Gata4, which have been shown to be expressed in TBX1-positive multipotent cardiac progenitor clones .

What flow cytometry parameters are optimal for detecting FITC-conjugated TBX1 antibody signal?

For optimal detection of FITC-conjugated TBX1 antibody in flow cytometry, the following parameters and considerations are recommended:

• Excitation/Emission settings:

  • Excitation: 488 nm laser (blue)

  • Emission filter: 530/30 nm bandpass filter

• Compensation settings:

  • FITC has potential spectral overlap with PE and other fluorophores

  • When multiplexing, use single-stained controls for each fluorophore to establish proper compensation matrix

  • Include FMO (Fluorescence Minus One) controls to set accurate gates

• Cell preparation protocol:

  • For intracellular TBX1 detection, use a fixation/permeabilization buffer with formaldehyde followed by methanol treatment or commercial kits designed for transcription factor staining

  • Blocking step with 2% FBS or BSA in PBS for 30 minutes at room temperature

  • For TBX1 detection in lymphatic endothelial cells, additional surface markers (e.g., LYVE-1, PROX1) may help in proper population gating

• Signal optimization:

  • Antibody titration is essential (typically 0.5-5 μg per million cells)

  • Incubation at 4°C for 30-45 minutes in the dark

  • Thorough washing to reduce background fluorescence

• Data analysis considerations:

  • Use appropriate negative controls (isotype control conjugated to FITC)

  • For quantification, mean fluorescence intensity (MFI) is more informative than percent positive when analyzing transcription factor expression levels

When investigating TBX1 in complex tissue like cardiac samples, consider additional markers for proper identification of cell subpopulations as demonstrated in lymphatic endothelial cell research .

How can ChIP-seq be performed using TBX1 antibodies to identify direct transcriptional targets?

ChIP-seq with TBX1 antibodies requires specific optimization for successful identification of transcriptional targets. Based on published research methodologies , the following protocol is recommended:

• Cross-linking and chromatin preparation:

  • Cross-link cells/tissues with 1% formaldehyde for 10 minutes at room temperature

  • Quench with 0.125M glycine for 5 minutes

  • Isolate nuclei and sonicate chromatin to 200-500 bp fragments

  • Verify fragment size by agarose gel electrophoresis

• Immunoprecipitation with TBX1 antibody:

  • Pre-clear chromatin with protein A/G beads

  • Incubate chromatin with 3-5 μg of TBX1 antibody overnight at 4°C

  • For FITC-conjugated antibodies, additional optimization may be required as the fluorophore could potentially interfere with antigen recognition

  • Include appropriate controls: IgG control and input DNA

• DNA purification and library preparation:

  • Reverse cross-links and purify DNA

  • Prepare libraries following standard NGS protocols with adapters

  • Validate libraries by qPCR for known TBX1 targets before sequencing

• Data analysis approach:

  • Map reads to reference genome

  • Call peaks using MACS2 or similar algorithms

  • Motif analysis to identify TBX1 binding consensus

  • Filter peaks through comparison with ATAC-seq data to identify accessible chromatin regions

Research has identified TBX1 binding to specific promoter regions, such as the miR-96 promoter where a binding site was found 3251 base pairs upstream of the transcription start site . TBX1 ChIP-seq analysis in cardiac tissue revealed binding to promoters of genes involved in immune tolerance, T cell activation, endothelial cell growth, and migration, with 23.1% of differentially expressed genes associated with TBX1 binding peaks .

What experimental approaches can distinguish between direct and indirect TBX1 transcriptional regulation?

Distinguishing between direct and indirect TBX1 transcriptional regulation requires multiple complementary experimental approaches:

• Integrated ChIP-seq and RNA-seq analysis:

  • Perform TBX1 ChIP-seq to identify binding sites

  • Conduct RNA-seq on TBX1 knockdown/knockout vs. control cells

  • Genes that show both TBX1 binding and differential expression are potential direct targets

  • Research has demonstrated that 23.1% of differentially expressed genes were associated with TBX1 binding peaks

• Reporter gene assays with mutational analysis:

  • Clone promoter regions containing putative TBX1 binding sites into luciferase reporter vectors

  • Test activity with wild-type TBX1 vs. domain-specific mutants

  • Perform site-directed mutagenesis of TBX1 binding sites

  • For example, research has shown that TBX1 directly represses the miR-96 promoter, with mutational analysis confirming functional regulation

• Electrophoretic Mobility Shift Assay (EMSA):

  • Verify direct DNA binding using purified TBX1 protein and labeled DNA probes

  • Include competition with unlabeled probes and supershift with TBX1 antibody

  • This approach has been used to confirm GATA6 binding to the TBX1 promoter

• Rapid response gene expression analysis:

  • Use inducible TBX1 expression systems combined with protein synthesis inhibitors

  • Monitor gene expression changes at early time points (1-4 hours)

  • Genes that respond rapidly are more likely direct targets

• CRISPRi targeting of specific binding sites:

  • Use CRISPR interference to specifically block TBX1 binding at individual sites

  • Measure the effect on target gene expression

  • This provides site-specific evidence for direct regulation

For example, studies have confirmed TBX1 direct regulation of Dtx1, a Notch E3 ligase that promotes lymphangiogenesis , and direct repression of miR-96 expression using these integrated approaches.

How can the TBX1 antibody be used to investigate protein-protein interactions in different developmental contexts?

TBX1 antibody can be used to investigate context-specific protein-protein interactions through multiple approaches:

• Co-immunoprecipitation (Co-IP) with TBX1 antibody:

  • Lyse cells or tissues from specific developmental stages

  • Immunoprecipitate using TBX1 antibody conjugated to beads

  • Identify interacting partners by western blot or mass spectrometry

  • Research has identified interactions between TBX1 and proteins like PITX2 and SRF

• Proximity ligation assay (PLA):

  • Use TBX1 antibody in combination with antibodies against suspected interacting proteins

  • This technique visualizes protein-protein interactions in situ with subcellular resolution

  • Particularly valuable for tissue sections from different developmental stages

• FRET or BRET analysis:

  • For live cell studies, combine fluorescently tagged TBX1 with potential partners

  • Measure energy transfer as indication of protein proximity

  • This provides dynamic interaction information in living cells

• Domain mapping experiments:

  • Use GST-tagged truncated TBX1 proteins to identify interaction domains

  • Research has shown that PITX2 binds to the N-terminus of TBX1, and this interaction is critical for repressing PITX2 transcriptional activity

  • The experimental approach involved generating a series of TBX1 truncated proteins (TBX1 FL, TBX1 ΔC, TBX1 ΔTC, TBX1 T-box, and TBX1 ΔNT) and testing their interaction with PITX2

• ChIP-reChIP (sequential ChIP):

  • First ChIP with TBX1 antibody followed by second ChIP with antibody against potential co-factor

  • This identifies genomic regions where both proteins are simultaneously bound

An example of interaction analysis is the study showing that the N-terminus of TBX1 interacts with PITX2 to repress PITX2 transcriptional activity . The researchers used GST-TBX1 pull-down experiments with different truncated TBX1 proteins to map the interaction domain.

What are common causes of false negative results when using TBX1 FITC-conjugated antibody?

Several factors can contribute to false negative results when using TBX1 FITC-conjugated antibody:

• Epitope masking due to fixation issues:

  • Overfixation with paraformaldehyde can mask TBX1 epitopes

  • Solution: Optimize fixation time (typically 10-15 minutes) or try antigen retrieval methods such as citrate buffer (pH 6.0) treatment for 10-20 minutes

• Inadequate permeabilization for nuclear antigen:

  • TBX1 is a nuclear transcription factor

  • Solution: Ensure sufficient permeabilization with 0.2-0.5% Triton X-100 for at least 15 minutes

• Developmental timing considerations:

  • TBX1 expression is highly stage-specific

  • Solution: Verify the developmental stage carefully; for example, TBX1 expression in the SHF changes significantly between 5-22 somites stages

• Tissue-specific expression levels:

  • TBX1 is expressed in specific cell populations like undifferentiated SHF cells and lymphatic endothelial cells

  • Solution: Include positive control tissues with known TBX1 expression

• Photobleaching of FITC fluorophore:

  • FITC is susceptible to photobleaching

  • Solution: Minimize exposure to light during processing, use anti-fade mounting media, and capture images promptly

• Antibody degradation:

  • FITC-conjugated antibodies can degrade over time

  • Solution: Store antibody according to manufacturer recommendations (typically at 4°C in the dark), aliquot to avoid freeze-thaw cycles

• Low expression levels:

  • TBX1 may be expressed at low levels in some contexts

  • Solution: Consider signal amplification methods such as tyramide signal amplification (TSA)

If false negative results persist, verify TBX1 expression using alternative methods such as RT-PCR for the transcript or using a different antibody that recognizes a different epitope.

How can researchers verify the specificity of TBX1 FITC-conjugated antibody staining?

Verifying the specificity of TBX1 FITC-conjugated antibody staining is crucial for accurate interpretation of results. The following validation approaches are recommended:

• Genetic controls:

  • Use Tbx1 knockout or knockdown samples as negative controls

  • Research has demonstrated loss of TBX1 staining in Tbx1-/- embryos

  • If working with a conditional knockout model such as Fabp4-Cre;Tbx1flox/flox, verify loss of staining in the specific cell population

• Peptide competition assay:

  • Pre-incubate TBX1 antibody with the immunizing peptide

  • This should abolish specific staining

• Multiple antibody validation:

  • Compare staining pattern with different TBX1 antibodies that recognize distinct epitopes

  • Consistent staining patterns increase confidence in specificity

• Correlation with mRNA expression:

  • Perform in situ hybridization for Tbx1 mRNA in parallel with immunostaining

  • Patterns should correlate closely

• Expected biological distribution validation:

  • Verify that staining patterns match known TBX1 expression patterns

  • For example, TBX1 should be restricted to undifferentiated SHF cells and not overlap with differentiation markers like α-SMA

• Western blot verification:

  • Confirm that the antibody recognizes a protein of the expected molecular weight

  • TBX1 is approximately 50 kDa

• Multiplexed staining analysis:

  • Co-stain with established markers of TBX1-expressing cells

  • In heart development, TBX1 should overlap with Isl1 in the SHF

Research has employed these validation approaches, for example showing that TBX1 and Isl1 co-staining patterns overlap in the SHF, while TBX1 and α-SMA show virtually no overlap, confirming TBX1's restriction to undifferentiated cells .

What quantitative methods are most reliable for analyzing TBX1 expression levels in tissue samples?

For reliable quantification of TBX1 expression in tissue samples, several methods with different strengths can be employed:

• Immunofluorescence quantification:

  • Measure mean fluorescence intensity in defined nuclear areas

  • Analyze at least 50-100 cells per sample for statistical validity

  • Controls: Include calibration standards with known fluorophore concentrations

  • Advantage: Provides spatial information and cell-specific expression

  • Example application: Quantifying TBX1 levels in specific regions of the SHF

• Flow cytometry analysis:

  • Measure mean fluorescence intensity (MFI) of FITC signal in TBX1+ cell populations

  • Use isotype controls to establish background levels

  • Analysis approach: Report both percentage of positive cells and MFI values

  • Advantage: High-throughput analysis of large cell numbers

  • Example application: Quantifying TBX1 levels in lymphatic endothelial cells

• Western blot densitometry:

  • Normalize TBX1 band intensity to housekeeping protein (β-actin, GAPDH)

  • Use calibration curves with recombinant protein standards

  • Controls: Include positive control samples with known TBX1 expression

  • Advantage: Assesses total protein levels in tissue samples

  • Example application: Comparing TBX1 levels between wild-type and mutant tissues

• RT-qPCR for transcript quantification:

  • Use validated TBX1-specific primers and probes

  • Normalize to multiple reference genes (at least 3) chosen for stability

  • Analysis: Apply ΔΔCt method with efficiency correction

  • Advantage: Highly sensitive detection of expression changes

  • Example application: Confirming changes in TBX1 expression in Tbx1 mutant studies

• Chromatin immunoprecipitation quantification (ChIP-qPCR):

  • Quantify TBX1 binding to specific genomic regions

  • Normalize to input DNA and IgG control

  • Example application: Quantifying TBX1 binding to the miR-96 promoter region

For the most reliable analysis, it is recommended to combine at least two independent quantification methods. For example, researchers have used both immunofluorescence and RT-PCR to verify TBX1 expression patterns in cardiac progenitor cells .

How can researchers distinguish between TBX1 isoforms using antibody-based detection methods?

Distinguishing between TBX1 isoforms requires careful selection of antibodies and experimental design:

• Isoform-specific antibody selection:

  • Choose antibodies raised against epitopes unique to specific isoforms

  • For FITC-conjugated antibodies, verify which isoform epitope is recognized

  • When isoform-specific antibodies are not available, complementary approaches are needed

• Western blot analysis for size discrimination:

  • Different TBX1 isoforms have distinct molecular weights

  • Use high-resolution gel systems (8-10% SDS-PAGE) for optimal separation

  • Include positive controls expressing specific isoforms

• Combined immunoprecipitation and mass spectrometry:

  • Immunoprecipitate TBX1 using a pan-TBX1 antibody

  • Identify isoform-specific peptides by mass spectrometry

  • This approach provides definitive isoform identification

• RT-PCR with isoform-specific primers:

  • Design primers spanning exon junctions specific to each isoform

  • Correlate protein detection with transcript expression

• Immunofluorescence combined with FISH:

  • Use fluorescence in situ hybridization with isoform-specific probes

  • Co-localize with TBX1 antibody staining

• Expression of tagged isoforms:

  • Generate cell lines expressing individually tagged TBX1 isoforms

  • Use as references for antibody validation and specificity

Research has shown that different TBX1 isoforms may have distinct functions. For example, when investigating TBX1 mutations associated with 22q11.2 deletion syndrome, researchers tested multiple TBX1 variant proteins (F148Y, H194Q, G310S) and found differential effects on transcriptional activity , suggesting functional differences between protein variants that might also apply to natural isoforms.

What are the implications of TBX1 subcellular localization patterns for interpreting function?

TBX1 subcellular localization provides important functional insights that should be considered when interpreting immunofluorescence data:

• Nuclear localization patterns:

  • As a transcription factor, TBX1 predominantly localizes to the nucleus in actively regulating cells

  • Punctate nuclear pattern may indicate association with transcriptional complexes

  • Changes in nuclear distribution can signal altered transcriptional activity

  • Research shows strong nuclear TBX1 staining in undifferentiated progenitor cells, with loss of expression upon differentiation

• Cytoplasmic localization:

  • Unexpected cytoplasmic TBX1 could indicate:
    a) Regulation of nuclear-cytoplasmic shuttling
    b) Post-translational modifications affecting localization
    c) Protein-protein interactions sequestering TBX1 outside the nucleus

  • Verify unexpected cytoplasmic staining with multiple antibodies

• Cell-type specific patterns:

  • TBX1 shows strong nuclear localization in multipotent progenitors but is absent in differentiated cells

  • Lymphatic endothelial cells show specific TBX1 expression patterns

  • Changes in localization patterns between cell types may indicate different functional roles

• Developmental dynamics:

  • TBX1 localization changes during development

  • In the SHF, TBX1 expression becomes more restricted as development proceeds

  • Temporal changes in localization should be interpreted in developmental context

• Quantitative approaches for localization analysis:

  • Nuclear/cytoplasmic ratio quantification

  • Co-localization with nuclear subcompartment markers

  • High-resolution imaging techniques like super-resolution microscopy

Functional implications of TBX1 localization have been demonstrated in research showing that TBX1 expression is restricted to undifferentiated progenitor cells, where it promotes proliferation and prevents premature differentiation . The disappearance of TBX1 from cells beginning to express differentiation markers indicates a functional switch in cellular programming.

How can RNA-seq data be integrated with TBX1 antibody studies to elucidate regulatory networks?

Integrating RNA-seq data with TBX1 antibody studies enables comprehensive mapping of TBX1-regulated networks:

• Differential expression analysis in TBX1 loss/gain of function models:

  • Compare transcriptomes of wild-type vs. TBX1 knockout/knockdown tissues

  • Analyze TBX1 overexpression models to identify repressed genes

  • Example approach: RNA-seq analysis of Fabp4-Cre;Tbx1flox/flox vs. control tissues identified differentially expressed genes involved in lymphangiogenesis and immunomodulation

• Integrative analysis with ChIP-seq data:

  • Overlay RNA-seq DEGs with TBX1 ChIP-seq peaks

  • Identify direct transcriptional targets showing both binding and expression changes

  • Research has shown that 23.1% of differentially expressed genes were associated with TBX1 binding peaks, including functionally relevant genes like Icam1 and Dtx1

• Time-course studies to capture dynamic regulation:

  • Perform RNA-seq at multiple time points following TBX1 manipulation

  • Identify immediate-early vs. secondary response genes

  • Construct temporal regulatory networks

• Single-cell approaches for cellular heterogeneity:

  • Combine scRNA-seq with TBX1 antibody-based cell sorting

  • Identify cell population-specific TBX1 regulatory networks

  • Example: Analysis of cardiac cell populations in Fabp4-Cre;Tbx1flox/flox hearts identified specific changes in immune cell composition, particularly increased T cell populations

• Pathway and network analysis tools:

  • Use tools like GSEA, IPA, or STRING for functional annotation

  • Identify enriched pathways in TBX1-regulated genes

  • Research has identified enrichment for genes involved in immune tolerance, autoimmune response, T cell activation, and endothelial cell processes

• Validation of key network nodes:

  • Select key genes from RNA-seq for detailed validation

  • Use TBX1 antibody for ChIP-qPCR confirmation of binding

  • Example: Confirmation of TBX1 binding to the miR-96 promoter followed by validation of functional regulation

An example of integrated analysis is the identification of Dtx1 as a TBX1 target gene through combined ChIP-seq and RNA-seq, with subsequent functional validation showing that reduced Dtx1 expression in TBX1-deficient cells leads to increased Notch1 and decreased VEGFR3, explaining the lymphangiogenic defects observed .

How can TBX1 antibodies be utilized in stem cell differentiation protocols for cardiac lineages?

TBX1 antibodies can enhance stem cell differentiation protocols for cardiac lineages in several ways:

• Real-time monitoring of differentiation states:

  • Use FITC-conjugated TBX1 antibodies in live cell imaging

  • Track the proportion of TBX1-positive progenitors during differentiation

  • Optimize culture conditions based on TBX1 expression dynamics

  • Research shows TBX1 marks multipotent cardiac progenitors that can differentiate into endothelial, smooth muscle, and cardiomyocyte lineages

• Purification of cardiac progenitor populations:

  • FACS-based isolation of TBX1-positive cardiac progenitors

  • Protocol details:
    a) Dissociate differentiating stem cell cultures at day 4-7
    b) Stain with FITC-conjugated TBX1 antibody (intracellular staining protocol)
    c) Sort TBX1+ cells for expansion or directed differentiation
    d) Expected yield: Typically 5-15% of cells from correctly staged cultures

• Quality control metrics for cardiac differentiation:

  • Quantitative assessment of TBX1-positive cells as progress indicator

  • Benchmark values based on research data:
    a) Day 4-5: 15-25% TBX1+ cells indicates proper SHF specification
    b) Day 7-8: Decreasing TBX1+ percentage indicates appropriate differentiation progression
    c) Terminal stages: Few to no TBX1+ cells in properly differentiated cultures

• Optimization of small molecule modulators:

  • Test different factors that regulate TBX1 expression

  • For example, modulating GATA6 expression could enhance TBX1 levels as research shows GATA6 activates TBX1 transcription

  • Assay optimization: Measure TBX1 expression using flow cytometry or immunofluorescence quantification

• Disease modeling applications:

  • Test the effect of TBX1 mutations associated with 22q11.2 deletion syndrome on cardiac differentiation

  • Research identified TBX1 variants in the cis-regulatory element that impaired GATA6-mediated transcriptional activation

  • Combine genome editing of TBX1 regulatory regions with antibody-based detection

Research has demonstrated that TBX1-expressing cells represent multipotent progenitors that can give rise to three heart lineages in clonal assays , making TBX1 antibody-based approaches valuable for generating specific cardiac populations.

What are the considerations for multiplexing TBX1 FITC antibody with other fluorescent markers?

Effective multiplexing of TBX1 FITC antibody with other fluorescent markers requires careful planning:

• Spectral compatibility considerations:

  • FITC emission spectrum (peak ~520 nm) overlaps partially with other green fluorophores

  • Optimal fluorophore combinations with FITC:
    a) DAPI (blue, nuclear counterstain)
    b) Cy3/TRITC/RFP (red)
    c) APC/Cy5/Alexa 647 (far red)

  • Avoid PE (yellow-orange) due to significant spectral overlap with FITC

• Staining protocol optimization:

  • Sequential staining approach:
    a) Perform TBX1 FITC staining first
    b) Block with excess unconjugated anti-mouse IgG if using other mouse antibodies
    c) Proceed with additional markers

  • Antibody concentrations may need adjustment in multiplex settings

• Controls for multiplexed experiments:

  • Single-stained controls for spectral compensation

  • Fluorescence minus one (FMO) controls to set accurate gates

  • Isotype controls for each fluorophore to assess background

• Biologically relevant co-staining combinations:

  • TBX1 + Isl1: For identifying cardiac progenitor populations

  • TBX1 + α-SMA: For distinguishing undifferentiated from differentiated cells

  • TBX1 + PECAM1/CD31: For endothelial lineage studies

  • TBX1 + Notch1 + VEGFR3: For investigating lymphatic endothelial regulation

• Imaging considerations:

  • Sequential scanning to minimize bleed-through

  • Post-acquisition linear unmixing for closely overlapping spectra

  • Consistent exposure settings for quantitative comparisons

• Flow cytometry panel design:

  • Place FITC-TBX1 on the 488 nm laser line

  • Adjust compensation settings using single-stained controls

  • Consider brightness hierarchy when designing panels (place dimmer markers on brighter fluorophores)

Research has successfully employed multiplexed approaches, such as co-staining for TBX1 with Isl1 to identify their overlapping expression patterns in the SHF, and TBX1 with α-SMA to demonstrate their mutually exclusive expression patterns .

How can TBX1 antibody studies contribute to understanding congenital heart defects?

TBX1 antibody studies provide critical insights into congenital heart defects (CHDs) through multiple research approaches:

• Spatial-temporal mapping of TBX1 expression in normal and pathological development:

  • Compare TBX1 expression patterns between normal and CHD models

  • Precise quantification of expression in specific cardiac regions

  • Research shows TBX1 is crucial for proper development of the second heart field (SHF) and outflow tract

• Analysis of TBX1 variants and regulatory mutations:

  • Assess the effect of pathogenic TBX1 mutations on protein localization and expression

  • Studies have identified rare TBX1 mutations within the cis-regulatory element in sporadic conotruncal heart defect patients

  • Functional analysis showed these variants impaired GATA6-mediated transcriptional activation of TBX1

• Investigation of molecular mechanisms:

  • Use TBX1 antibodies to study protein interactions disrupted in CHD

  • ChIP studies to identify altered genomic binding in mutant models

  • Research demonstrated that TBX1 regulates cardiac progenitor cell proliferation and differentiation, with loss of TBX1 causing premature differentiation

• Lineage tracing combined with phenotypic analysis:

  • Track the fate of TBX1-expressing cells in normal and mutant hearts

  • Correlate cell distribution anomalies with structural defects

  • Clonal analysis has shown TBX1-expressing cells contribute to multiple cardiac lineages

• Molecular signatures of TBX1-associated CHD:

  • Compare transcriptional profiles of TBX1-positive cells in normal vs. CHD models

  • Identify downstream effectors as potential therapeutic targets

  • Research identified TBX1 regulation of genes involved in cardiovascular development including VEGFR3 and DLL4 in brain endothelial cells

• Integration with human genetic studies:

  • Correlate antibody-based findings in model systems with human mutations

  • TBX1 is the major gene involved in 22q11.2 deletion syndrome, a common genetic cause of CHD

  • Functional studies of human mutations (F148Y, H194Q, G310S) have shown differential effects on transcriptional activity