SCN10A Antibody, FITC conjugated

What is SCN10A and why is it significant in research?

SCN10A encodes the Nav1.8 voltage-gated sodium channel, which plays critical roles in neuronal excitability and action potential generation. It is classified as a tetrodotoxin (TTX)-resistant sodium channel, primarily expressed in dorsal root ganglion (DRG) neurons . Recent research has revealed that SCN10A also has significant cardiac functions, with implications for conduction disorders and arrhythmia prevention . The protein is essential for the generation of action potentials and cell excitability, activated in response to depolarization to selectively allow sodium ion flow . SCN10A's dual role in neuronal and cardiac tissues makes it an important target for both pain research and cardiac electrophysiology studies.

What are the primary applications for fluorescently-labeled SCN10A antibodies?

Fluorescently-labeled SCN10A antibodies are primarily used for immunofluorescence (IF), immunocytochemistry (ICC), and flow cytometry (FC) applications . In neurological research, these antibodies enable visualization of SCN10A/Nav1.8 expression patterns in DRG neurons and help determine co-localization with other neuronal markers such as synaptophysin and TRPV1 . In cardiac research, they can be used to confirm overexpression of SCN10A gene therapy constructs, as demonstrated in studies examining the therapeutic potential of SCN10A-short (S10s) transcripts . The fluorescent conjugation eliminates the need for secondary antibodies, simplifying multiplexing experiments and reducing background signal.

How does SCN10A-short (S10s) differ from full-length SCN10A?

SCN10A-short (S10s) is a truncated transcript that encodes only the carboxy-terminal domain of the full-length neuronal sodium channel . Unlike the complete SCN10A protein, which forms a functional voltage-gated sodium channel, S10s appears to modulate the function of other sodium channels, particularly Nav1.5 (encoded by SCN5A). Recent research demonstrates that S10s overexpression increases cellular sodium current (INa) and enhances action potential characteristics including maximal upstroke velocity (dV/dtmax) and amplitude in cardiomyocytes . This modulation occurs without significantly affecting resting membrane potential or action potential duration, suggesting S10s influences channel availability rather than gating kinetics. This unique property makes S10s particularly valuable as a potential gene therapy target for treating conduction disorders caused by reduced sodium current.

What are the recommended storage conditions for FITC-conjugated antibodies?

Fluorescently-conjugated antibodies, including FITC-conjugated SCN10A antibodies, require specific storage conditions to maintain functionality and fluorescence intensity. These antibodies should typically be stored at -20°C in the dark to prevent photobleaching of the fluorophore. For antibodies intended for flow cytometry applications, a concentration of approximately 0.40 μg per 10^6 cells is recommended . When using these antibodies for immunofluorescence applications, proper titration is essential, with recommended dilutions typically ranging from 1:10 to 1:100 depending on the specific application and sample type . It's important to note that all fluorescently-labeled antibodies should be protected from prolonged light exposure during storage and experimental procedures to preserve signal intensity.

What are the optimal fixation and permeabilization protocols for SCN10A immunostaining?

For optimal SCN10A immunostaining, fixation and permeabilization protocols must be carefully selected based on the subcellular localization of the target epitope. The antibody described in the search results targets an intracellular C-terminus epitope (amino acid residues 1943-1956 of rat Nav1.8) , requiring appropriate cell permeabilization. For immunofluorescence applications in tissue sections or cultured cells, a recommended protocol includes:

• Fixation with 4% paraformaldehyde for 15-20 minutes at room temperature

• Washing steps with PBS (3 × 5 minutes)

• Permeabilization with 0.2-0.3% Triton X-100 in PBS for 10 minutes

• Blocking with 5-10% normal serum (from the species unrelated to the primary antibody) for 1 hour

• Incubation with the fluorescently-labeled SCN10A antibody at dilutions between 1:10 and 1:100

This protocol has been successfully demonstrated in immunohistochemical staining of rat DRG sections, where Nav1.8 labeling appears predominantly in cell bodies rather than nerve fibers . For flow cytometry applications, standard cell fixation and permeabilization methods appropriate for intracellular staining should be employed, with antibody concentrations of approximately 0.40 μg per 10^6 cells .

How can I design multiplex immunofluorescence experiments with SCN10A antibodies?

Designing effective multiplex immunofluorescence experiments with SCN10A antibodies requires careful selection of complementary fluorophores and consideration of expression patterns. Based on successful multiplex stainings documented in the search results:

• Selection of compatible fluorophores: When using a FITC-conjugated SCN10A antibody, complementary choices include red-spectrum dyes (such as ATTO-594 or Cy5) for co-staining proteins of interest. This prevents spectral overlap while allowing distinct visualization of different targets.

• Validation of co-expression patterns: Previous research has demonstrated partial co-localization of Nav1.8 with both synaptophysin and TRPV1 in DRG neurons . When designing new multiplex experiments, consider known expression patterns to formulate biologically relevant hypotheses.

• Nuclear counterstaining: DAPI counterstaining provides valuable context for cellular localization, as demonstrated in the multiplex staining of rat DRG samples .

• Controls and antibody validation: Include appropriate single-stain controls to verify specificity and rule out cross-reactivity or spectral bleed-through.

This approach has been validated in studies examining the co-localization of Nav1.8 with synaptophysin and TRPV1, revealing partial overlap in their distribution within DRGs . Such multiplex experiments are particularly valuable for studying the functional relationships between ion channels and other neuronal proteins.

What controls should be included when using fluorescently-labeled SCN10A antibodies?

When designing experiments with fluorescently-labeled SCN10A antibodies, several controls are essential to ensure result validity:

• Negative controls:

  • Isotype control: Use an isotype-matched rabbit IgG conjugated to the same fluorophore at equivalent concentration

  • No primary antibody control: Apply only blocking solution without antibody to assess autofluorescence

  • Absorption control: Pre-incubate the antibody with its immunizing peptide to verify specificity

• Positive controls:

  • Known positive samples: Include tissues or cell lines with confirmed SCN10A expression (e.g., DRG neurons for Nav1.8 or SH-SY5Y cells that have been validated as positive )

  • Counterstaining with established markers: For DRG studies, include markers like TRPV1 that show partial co-localization

• Technical controls:

  • Single-color controls: In multiplex experiments, include samples stained with each fluorophore individually

  • Concentration gradients: Test a range of antibody dilutions (1:10 to 1:100 for IF/ICC ) to determine optimal signal-to-noise ratio

These controls help distinguish specific from non-specific staining and ensure proper interpretation of results, particularly important when evaluating the subcellular localization patterns of Nav1.8, which has been observed predominantly in cell bodies rather than nerve fibers in DRG neurons .

How can SCN10A antibodies be used to validate gene therapy approaches?

Fluorescently-labeled SCN10A antibodies serve as crucial validation tools for gene therapy approaches targeting sodium channel modulation. In recent research investigating SCN10A-short (S10s) as a gene therapy candidate, antibodies were instrumental in confirming successful transgene expression . The process involves:

• Transduction verification: Following administration of viral vectors (such as AAV6 or AAV9) expressing S10s, immunofluorescence staining can confirm the presence of the transgene product in target tissues. This verification is critical before proceeding to functional assessments.

• Expression pattern analysis: Antibody staining helps determine the spatial distribution of transgene expression throughout cardiac tissue, confirming targeted delivery to cardiomyocytes versus non-specific expression.

• Quantitative assessment: Beyond qualitative confirmation, antibody staining can be used to estimate transduction efficiency by determining the percentage of cells expressing the transgene.

• Co-localization studies: Multiplexed staining with channel subunit-specific antibodies can elucidate potential interactions between the therapeutic transgene and endogenous sodium channels.

Recent studies have successfully used this approach to confirm S10s overexpression in mouse heart tissue following intramyocardial and intravenous administration of AAV vectors, demonstrating the utility of antibody validation in gene therapy research .

What are the implications of SCN10A modulation for cardiac arrhythmia research?

Recent research has revealed significant implications of SCN10A modulation for cardiac arrhythmia treatment, with antibodies playing a crucial role in mechanistic studies. The findings demonstrate:

• Enhanced sodium current: SCN10A-short (S10s) gene therapy increases cellular sodium current (INa) in cardiomyocytes, as verified through patch-clamp studies of isolated cells from treated animals .

• Improved conduction velocity: Optical mapping studies, complemented by immunofluorescence confirmation of transgene expression, have shown that S10s gene therapy rescues conduction slowing in Scn5a-haploinsufficient mice, a model of cardiac conduction disorders .

• Arrhythmia prevention: Beyond improving baseline conduction, S10s overexpression prevents ventricular tachycardia induced by ischemia-reperfusion in wild-type mice, suggesting broader applications in preventing acquired arrhythmias .

• Translational potential: Studies in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) demonstrate that S10s increases action potential upstroke velocity, suggesting therapeutic potential in human cardiac disorders .

These findings have significant implications for treating sodium channelopathies, particularly Brugada syndrome and progressive cardiac conduction disease, which result from SCN5A haploinsufficiency and reduced sodium current. The ability to track transgene expression using antibodies provides critical validation for these therapeutic approaches.

How can co-localization studies with SCN10A inform our understanding of pain pathways?

Co-localization studies using fluorescently-labeled SCN10A antibodies provide valuable insights into pain signaling pathways by revealing functional relationships between Nav1.8 and other neuronal proteins. Key findings and methodological approaches include:

• SCN10A and TRPV1 partial co-localization: Immunohistochemical studies using multiplex staining have demonstrated partial co-localization between Nav1.8 and TRPV1 in dorsal root ganglion (DRG) neurons . This relationship is physiologically significant as both channels contribute to nociception, with TRPV1 serving as a thermal pain sensor and Nav1.8 propagating action potentials in pain-sensing neurons.

• Synaptophysin relationship: Multiplex staining has also revealed partial overlap in the distribution of Nav1.8 and synaptophysin within DRGs . As synaptophysin is a marker for synaptic vesicles, this co-localization suggests potential involvement of Nav1.8 in regulating neurotransmitter release at synapses.

• Methodological considerations: These co-localization studies typically employ:

  • Multiple fluorophores (e.g., ATTO Fluor-594 for Nav1.8 and ATTO Fluor-488 for TRPV1)

  • Confocal microscopy for precise spatial resolution

  • DAPI counterstaining to provide cellular context

The partial rather than complete overlap between Nav1.8 and these proteins suggests functional specialization within subpopulations of DRG neurons, potentially explaining the complex and heterogeneous nature of pain signaling. These findings help identify specific neuronal subtypes that may be targeted for pain therapies with improved specificity and reduced side effects.

What are common challenges in flow cytometry applications with SCN10A antibodies?

Flow cytometry with SCN10A antibodies presents several technical challenges that researchers should anticipate and address:

• Intracellular epitope accessibility: Since the antibody targets the intracellular C-terminus of Nav1.8 , proper fixation and permeabilization are crucial. Insufficient permeabilization can lead to false negatives, while excessive treatment may compromise cellular integrity and increase non-specific binding.

• Low expression levels: In cells with modest SCN10A expression, signal detection may be challenging. For optimal results:

  • Use the recommended concentration of 0.40 μg per 10^6 cells

  • Consider signal amplification methods if necessary

  • Ensure proper instrument calibration with appropriate voltage settings

• Autofluorescence interference: Particularly problematic when using FITC conjugates, as cellular autofluorescence often overlaps with FITC emission spectra. Strategies to address this include:

  • Including unstained controls to establish autofluorescence baseline

  • Using alternative fluorophores with emission in different spectral regions

  • Implementing autofluorescence compensation in analysis

• Cross-reactivity concerns: Validate antibody specificity through appropriate negative controls and positive controls using cell lines with confirmed reactivity, such as HeLa cells for flow cytometry applications .

For reliable results, follow validated protocols for intracellular staining and include both negative controls (isotype or secondary only) and positive control samples with known SCN10A expression, titrating the antibody to determine optimal concentration for your specific experimental system .

How can I optimize detection of low-abundance SCN10A expression in tissue samples?

Detecting low-abundance SCN10A expression in tissue samples requires optimization strategies that enhance signal while minimizing background:

• Antibody concentration optimization:

  • Begin with the recommended dilution range (1:10-1:100 for IF/ICC )

  • Perform systematic titration experiments to determine optimal concentration for your specific tissue

  • Consider extended incubation times (overnight at 4°C) to improve antigen binding

• Signal amplification methods:

  • For non-conjugated primary antibodies, employ tyramide signal amplification (TSA)

  • Use high-sensitivity detection systems with brighter fluorophores

  • Consider secondary antibody approaches if direct conjugates yield insufficient signal

• Background reduction strategies:

  • Implement stringent blocking protocols with 5-10% normal serum

  • Include 0.1-0.3% Triton X-100 in antibody diluents to reduce non-specific binding

  • Increase washing duration and frequency after antibody incubation

• Tissue-specific considerations:

  • For cardiac tissue, where SCN10A expression may be lower than in DRG, antigen retrieval methods may improve epitope accessibility

  • In gene therapy validation studies, compare treated tissues with controls to accurately detect increased expression

These approaches have proven effective in detecting SCN10A-short expression in cardiac tissues following gene therapy, even at intermediate viral doses where expression levels are relatively low .

What factors affect the specificity of SCN10A antibodies across different species?

When using SCN10A antibodies across different species, several factors influence specificity and must be carefully considered:

• Epitope conservation:

  • The epitope targeted by the antibody in search result corresponds to amino acid residues 1943-1956 of rat Nav1.8 (Accession Q63554)

  • Cross-reactivity depends on sequence conservation at this specific region across species

  • Human SCN10A shows high but not complete homology to rat sequences, potentially affecting binding affinity

• Antibody validation status:

  • Some SCN10A antibodies have only been validated in specific species (e.g., human samples for the antibody in search result )

  • Always verify whether the antibody has been tested in your species of interest

  • When using in non-validated species, include appropriate positive and negative controls

• Isoform specificity:

  • Different splice variants of SCN10A exist, including the SCN10A-short discussed in cardiac research

  • Verify whether the antibody recognizes all relevant isoforms in your experimental system

  • For studies involving SCN10A-short specifically, confirm the epitope location relative to the truncated protein

• Detection method considerations:

  • Sensitivity requirements may vary between species due to different expression levels

  • Optimization of antibody concentration may be necessary when transitioning between species

  • Background levels and non-specific binding can vary significantly between tissues from different species

When adapting protocols between species, always perform preliminary validation experiments to confirm specificity and optimize conditions for the new species, particularly when transitioning between rodent models and human samples.

How does SCN10A-short gene therapy compare to other approaches for treating cardiac conduction disorders?

SCN10A-short (S10s) gene therapy represents an innovative approach to treating cardiac conduction disorders, with distinct advantages compared to conventional treatments:

• Mechanism comparison:

  • Unlike antiarrhythmic drugs that often have multiple, sometimes opposing effects on different ion channels, S10s specifically increases sodium current (INa) without significantly affecting other electrophysiological parameters

  • Compared to pacemaker implantation, S10s gene therapy addresses the underlying pathophysiology by rescuing conduction velocity rather than bypassing the conduction system

  • Unlike full-length sodium channel gene therapy, S10s is significantly smaller, making it more amenable to packaging in adeno-associated viral (AAV) vectors

• Efficacy profile:

  • S10s gene therapy rescues conduction slowing in Scn5a-haploinsufficient mice, a model of cardiac conduction disorders

  • It prevents ventricular tachycardia induced by ischemia-reperfusion in wild-type mice

  • Computer simulations predict that S10s can restore conduction velocity to near-normal levels even in SCN5A haploinsufficiency

• Translational potential:

  • Studies in human induced pluripotent stem cell-derived cardiomyocytes confirm that S10s increases action potential upstroke velocity

  • In silico studies in simulated human ventricular tissue demonstrate potential efficacy across a range of expression levels

The ability to increase sodium current without requiring full-length channel expression represents a significant advantage for gene therapy approaches targeting conduction disorders, particularly for conditions like Brugada syndrome or progressive cardiac conduction disease caused by SCN5A haploinsufficiency.

What are the implications of SCN10A expression in non-neuronal tissues?

While traditionally studied in dorsal root ganglia neurons, emerging research has revealed significant SCN10A expression and function in non-neuronal tissues, particularly the heart, with important implications:

• Cardiac expression patterns:

  • SCN10A expression in cardiac tissue contributes to normal cardiac conduction

  • The carboxy-terminal domain of SCN10A (S10s) can modulate the function of cardiac sodium channels (Nav1.5), enhancing sodium current and action potential characteristics

  • This cross-regulation between neuronal and cardiac sodium channels represents an important mechanism for fine-tuning cardiac excitability

• Pathophysiological relevance:

  • Genome-wide association studies have linked SCN10A variants to cardiac conduction parameters and arrhythmia susceptibility

  • S10s gene therapy can rescue conduction defects in mouse models of sodium channelopathies and prevent arrhythmias induced by ischemia-reperfusion

  • These findings suggest that SCN10A plays a more significant role in cardiac electrophysiology than previously recognized

• Methodological considerations for studying non-neuronal expression:

  • Detection may require more sensitive methods due to potentially lower expression levels compared to DRG neurons

  • Functional studies should complement immunolocalization to confirm physiological relevance

  • Computer modeling can help predict the impact of altered SCN10A function on tissue-level electrophysiology

The expanding recognition of SCN10A's role beyond sensory neurons opens new avenues for understanding and treating both cardiac arrhythmias and pain disorders, highlighting the importance of integrated approaches to studying ion channel function across different tissues.

How do computational models inform our understanding of SCN10A modulation in cardiac tissue?

Computational modeling has become an invaluable tool for understanding the complex effects of SCN10A modulation in cardiac tissue, complementing experimental approaches with antibodies and electrophysiology:

• Prediction of therapeutic efficacy:

  • In silico experiments using computer-simulated human ventricular cardiomyocytes have demonstrated that SCN10A-short (S10s) can effectively restore conduction velocity in SCN5A haploinsufficiency models

  • These models show that even at reduced S10s expression levels (0.5× overexpression), significant improvement in conduction velocity can be achieved

  • Computational approaches allow systematic exploration of various expression levels and gap junction conductances that would be impractical to test experimentally

• Translation between species:

  • Models help bridge findings between mouse models and human applications

  • Simulations of human ventricular tissue incorporating data from isolated murine cardiomyocytes predict that S10s gene therapy could reduce conduction velocity deficits from 17-22% to just 3-4% in human SCN5A haploinsufficiency

• Mechanistic insights:

  • Computational models reveal that S10s not only improves conduction velocity but also enhances tissue excitability

  • The mutation-induced increase in stimulus current threshold is reduced from approximately 15% to just 1% with S10s therapy

  • These findings suggest multiple mechanisms through which S10s may prevent arrhythmias

These computational approaches provide crucial insights for translational research, helping to predict human outcomes based on preclinical data and optimize gene therapy approaches before clinical testing. When combined with experimental validation using antibodies to confirm expression patterns, computational modeling creates a powerful framework for advancing cardiac electrophysiology research.

What emerging technologies might enhance SCN10A research in the future?

Several emerging technologies hold promise for advancing SCN10A research beyond current antibody-based approaches:

• CRISPR-based tagging and visualization:

  • Endogenous tagging of SCN10A with fluorescent proteins or epitope tags could enable live-cell imaging without antibodies

  • This approach would overcome potential specificity issues associated with antibodies while allowing dynamic studies of channel trafficking and localization

• Super-resolution microscopy:

  • Techniques like STORM, PALM, and STED microscopy could reveal nanoscale distribution patterns of SCN10A in relation to other channel components

  • These approaches would provide unprecedented insights into channel clustering and interactions with regulatory proteins at the molecular level

• Optogenetic and chemogenetic modulation:

  • Light-activated or designer drug-activated sodium channels could enable precise temporal control of SCN10A activity

  • This would facilitate studies of how acute modulation of sodium currents affects cellular excitability and tissue-level conduction

• Single-cell transcriptomics and proteomics:

  • Integration of SCN10A expression data with comprehensive single-cell profiles would reveal co-expression patterns and potential regulatory networks

  • This could identify new therapeutic targets and biomarkers for sodium channelopathies

• Advanced computational modeling:

  • Building on existing work , multiscale models integrating molecular dynamics, cellular electrophysiology, and tissue-level conduction could provide more comprehensive understanding of SCN10A function

  • These models could guide personalized therapeutic approaches based on patient-specific genetic variants

These technologies, when combined with traditional antibody-based methods, will provide a more comprehensive understanding of SCN10A function in both normal physiology and disease states.

What are the current limitations in our understanding of SCN10A function?

Despite significant advances in SCN10A research, several important limitations remain in our understanding:

• Tissue-specific regulatory mechanisms:

  • While SCN10A is expressed in both neuronal and cardiac tissues, the mechanisms regulating its expression and function across different tissues remain incompletely understood

  • The differential effects of SCN10A modulation in neurons versus cardiomyocytes require further investigation to develop targeted therapeutic approaches

• Isoform-specific functions:

  • The distinct roles of full-length SCN10A versus SCN10A-short (S10s) in different physiological contexts need further clarification

  • Current antibody-based approaches may not always distinguish between these isoforms, potentially obscuring important functional differences

• Interactions with other sodium channel subunits:

  • The molecular mechanisms by which S10s modulates sodium current, particularly its interactions with Nav1.5 in cardiac tissue, remain incompletely characterized

  • Whether similar interactions occur with other sodium channel α-subunits is an important unanswered question

• Long-term effects of therapeutic modulation:

  • While short-term benefits of S10s gene therapy have been demonstrated in mouse models , potential long-term consequences of sustained SCN10A modulation require further investigation

  • Compensatory changes in other ion channels or regulatory mechanisms may occur with chronic modulation

• Human translational challenges:

  • Despite promising results in animal models and human cell lines, significant translational challenges remain in developing SCN10A-based therapies for human diseases

  • Individual variation in SCN10A expression and function may necessitate personalized approaches

Addressing these limitations will require integrated approaches combining advanced imaging, electrophysiology, molecular biology, and computational modeling to develop a more comprehensive understanding of SCN10A biology.

How might SCN10A research impact personalized medicine for pain and cardiac disorders?

SCN10A research is poised to make significant contributions to personalized medicine approaches for both pain management and cardiac disorders:

• Genetic variant-informed therapy:

  • Genome-wide association studies have linked SCN10A variants to both cardiac conduction parameters and pain sensitivity

  • Characterizing the functional consequences of these variants could enable genotype-guided therapy selection

  • Patients with specific SCN10A variants might benefit from targeted approaches like S10s gene therapy for cardiac conduction disorders

• Biomarker development:

  • Antibody-based detection of SCN10A expression patterns or post-translational modifications could serve as biomarkers for disease progression or treatment response

  • These biomarkers could help stratify patients for clinical trials and guide therapeutic decision-making

• Precision targeting of therapy:

  • Understanding the tissue-specific and subcellular distribution of SCN10A (as revealed by immunofluorescence studies ) could enable more precise therapeutic targeting

  • For pain management, therapies could selectively target SCN10A in nociceptive neurons while sparing cardiac expression

  • For cardiac disorders, approaches like S10s gene therapy could be delivered specifically to cardiac tissue

• Combination therapy optimization:

  • Knowledge of SCN10A interactions with other channels and receptors (such as TRPV1 ) could inform rational combination therapies

  • Computer modeling approaches could predict optimal combinations for individual patients based on their specific genetic profiles