SLC19A3 Antibody, FITC conjugated

What is SLC19A3 and why is it significant in neurological research?

SLC19A3 (Solute Carrier Family 19 Member 3) functions as a high-affinity thiamine transporter, mediating thiamine uptake via a proton anti-port mechanism. Unlike its family member SLC19A2, it has no folate transport activity but does facilitate H⁺-dependent pyridoxine transport . The protein has particular significance in neurological research because mutations in the SLC19A3 gene result in thiamine metabolism dysfunction syndrome 2, also known as biotin-thiamine-responsive basal ganglia disease (BTBGD) .

In the central nervous system, SLC19A3 expression is distinctively restricted to blood vessels, specifically localized at the basement membrane and within perivascular pericytes, whereas SLC19A2 is found at the luminal side of endothelial cells . This polarized distribution suggests both transporters are required for thiamine transport across the blood-brain barrier, explaining why SLC19A3 mutations primarily manifest as neurological symptoms despite normal systemic thiamine levels .

Polyclonal SLC19A3 antibodies, such as the FITC-conjugated variants described in the search results, recognize multiple epitopes on the SLC19A3 protein. For instance, the antibody described in result targets amino acids 217-246 from the central region of human SLC19A3. This multi-epitope recognition potentially provides greater sensitivity when detecting native proteins in complex matrices .

Monoclonal antibodies, by contrast, recognize a single epitope, offering higher specificity but potentially lower sensitivity. The choice between polyclonal and monoclonal depends on the research objectives:

• Polyclonal antibodies are preferable for:

  • General protein detection across multiple species (human, mouse, rat)

  • Applications requiring signal amplification

  • Detection of proteins in their native conformation

• Monoclonal antibodies excel in:

  • Distinguishing closely related protein isoforms

  • Reproducibility across experiments

  • Applications requiring minimal batch-to-batch variation

For SLC19A3 research, polyclonal antibodies like those described in the search results offer versatility across applications including Western blotting, immunofluorescence, and ELISA .

What methodological approaches optimize SLC19A3 detection in neural tissue samples?

Optimizing SLC19A3 detection in neural tissues requires careful consideration of the protein's localization pattern. Since SLC19A3 is primarily expressed in blood vessels rather than neuronal bodies, the following methodological considerations are crucial:

• Tissue preparation:

  • Perfusion fixation is recommended to preserve vascular structures

  • 4% formaldehyde fixation followed by 0.2% Triton X-100 permeabilization has been validated

  • Cryosections (10-20μm) are preferred over paraffin-embedded sections

• Antibody application:

  • Pre-blocking with 5-10% normal serum from the secondary antibody host species

  • Incubation with primary FITC-conjugated SLC19A3 antibody at 1:50-1:200 dilution

  • Extended incubation times (overnight at 4°C) to enhance signal specificity

• Signal detection:

  • Direct visualization of FITC signal (excitation ~495nm, emission ~519nm)

  • Nuclear counterstaining with DAPI for structural context

  • Co-staining with vascular markers (CD31, von Willebrand factor) for co-localization studies

For quantitative analyses, confocal microscopy with z-stack acquisition provides superior resolution of SLC19A3's perivascular localization pattern.

How can researchers validate SLC19A3 antibody specificity and overcome cross-reactivity challenges?

Antibody validation is crucial for ensuring reliable experimental outcomes, particularly for SLC19A3 where specificity concerns may arise due to homology with other family members (SLC19A1 and SLC19A2). Recommended validation strategies include:

• Positive controls:

  • HepG2 and SH-SY5Y cell lines demonstrate reliable endogenous expression

  • Western blotting should reveal a protein band at the predicted molecular weight of 56 kDa

• Negative controls:

  • Secondary antibody-only controls

  • Pre-absorption of antibody with immunizing peptide

  • SLC19A3 knockout or knockdown samples when available

• Cross-reactivity assessment:

  • Parallel testing with independently raised antibodies targeting different epitopes

  • Comparative staining patterns with SLC19A2 antibodies to confirm distinct localization

For FITC-conjugated antibodies specifically, autofluorescence controls are essential, particularly in neural tissues where lipofuscin can generate false-positive signals.

What are the critical considerations when designing co-localization experiments with SLC19A3 FITC-conjugated antibodies?

Co-localization studies investigating SLC19A3 interactions with other proteins require careful experimental design:

• Fluorophore selection:

  • When using FITC-conjugated SLC19A3 antibodies, complementary fluorophores for co-staining should have minimal spectral overlap

  • Recommended combinations: FITC (green) + Cy3 (red) or Alexa 594 (red)

• Sequential staining protocol:

  • First apply the non-conjugated primary antibody

  • Follow with its corresponding secondary antibody

  • Block any remaining secondary antibody binding sites

  • Apply the FITC-conjugated SLC19A3 antibody last

• Acquisition parameters:

  • Channel sequential scanning to minimize bleed-through

  • Identical acquisition settings across experimental groups

  • Z-stack acquisition for 3D co-localization assessment

• Quantitative analysis:

  • Pearson's correlation coefficient or Mander's overlap coefficient

  • Object-based co-localization for punctate structures

  • Distance-based analyses for proximal but non-overlapping signals

For blood-brain barrier studies specifically, triple labeling with SLC19A3 (FITC), SLC19A2, and endothelial/pericyte markers can reveal the polarized distribution described in the literature .

How can researchers utilize SLC19A3 antibodies to investigate thiamine transport dysregulation in disease models?

SLC19A3 antibodies provide valuable tools for investigating thiamine transport dysfunction in various disease models:

• Cellular models:

  • SLC19A3 expression analysis in cells exposed to thiamine deficiency conditions

  • Localization studies in cells expressing disease-associated SLC19A3 variants

  • Co-localization with autophagic or stress markers to assess cellular responses

• Methodological approach:

  • Immunofluorescence with FITC-conjugated SLC19A3 antibodies at 1:50-1:100 dilution

  • Western blotting (1:500 dilution) for quantitative expression analysis

  • Subcellular fractionation followed by immunodetection to assess membrane vs. cytoplasmic distribution

• Comparative analysis framework:

  • Wild-type vs. mutant SLC19A3 localization

  • Basal vs. thiamine-supplemented conditions

  • Presence vs. absence of biotin supplementation

For disease models of biotin-thiamine-responsive basal ganglia disease, antibodies targeting amino acids 217-246 (central region) or 150-300 of human SLC19A3 have been validated and can detect both wild-type and most mutant variants of the protein.

What methodological considerations are important when using SLC19A3 antibodies in blood-brain barrier research?

The unique polarized distribution of SLC19A3 at the blood-brain barrier presents specific methodological considerations:

• Sample preparation:

  • Fresh-frozen rather than fixed samples for quantitative studies

  • Micro-dissection of brain vascular structures for enriched analysis

  • Careful separation of parenchymal and vascular fractions

• Analytical approach:

  • Co-immunostaining with FITC-conjugated SLC19A3 antibodies (1:50-1:200) and markers such as:

    • Basement membrane: Collagen IV, laminin

    • Pericytes: PDGFRβ, NG2

    • Endothelial cells: CD31, claudin-5

  • Confocal microscopy with high numerical aperture objectives (≥1.3 NA)

  • Super-resolution techniques for precise localization

• Functional correlation:

  • Parallel assessment of thiamine transport activity

  • Correlation of SLC19A3 expression patterns with blood-brain barrier integrity markers

  • Comparison between brain regions with differential barrier properties

These approaches can help elucidate how SLC19A3 contributes to the polarized transport of thiamine across the blood-brain barrier, which is critical for understanding the neurological manifestations of SLC19A3 mutations .

What analytical techniques provide robust quantification of SLC19A3 expression using FITC-conjugated antibodies?

Quantitative analysis of SLC19A3 expression using FITC-conjugated antibodies requires standardized approaches:

• Imaging-based quantification:

  • Standardized acquisition parameters (exposure time, gain, offset)

  • Background subtraction and threshold-based segmentation

  • Integrated density measurements normalized to cell count or tissue area

  • 3D volume measurements for z-stack data

• Flow cytometry analysis:

  • Single-cell suspensions from tissues or cultured cells

  • Proper compensation controls for multiparameter analysis

  • Gating strategies that account for autofluorescence

  • Mean fluorescence intensity (MFI) comparison across experimental groups

• Plate-based fluorescence assays:

  • In-cell ELISA with FITC-conjugated SLC19A3 antibodies

  • Fluorescence intensity normalized to cell number (via DNA stain)

  • Standard curves using recombinant SLC19A3 protein if available

Regardless of the analytical approach, inclusion of appropriate controls (positive, negative, isotype) and standardization across experimental batches are essential for reliable quantification.

How can SLC19A3 antibodies contribute to understanding the pathophysiology of biotin-thiamine-responsive basal ganglia disease?

FITC-conjugated SLC19A3 antibodies offer unique insights into the pathophysiology of biotin-thiamine-responsive basal ganglia disease (BTBGD):

• Mutation impact assessment:

  • Immunolocalization studies comparing wild-type and mutant SLC19A3 trafficking

  • Co-localization with ER or Golgi markers to assess retention of mutant proteins

  • Quantitative analysis of membrane vs. cytoplasmic distribution

• Neuropathological investigations:

  • Analysis of SLC19A3 expression in post-mortem brain tissues from BTBGD patients

  • Correlation of SLC19A3 distribution with regions of neurodegeneration

  • Assessment of vascular SLC19A3 expression in affected basal ganglia regions

• Treatment response mechanisms:

  • Evaluation of SLC19A3 expression following biotin and/or thiamine supplementation

  • Investigation of biotin's role in SLC19A3 transcriptional regulation

  • Analysis of compensatory changes in SLC19A2 expression with thiamine treatment

These approaches can help elucidate why, despite having normal blood thiamine levels, BTBGD patients develop neurological symptoms and exhibit reduced free-thiamine levels in cerebrospinal fluid .

What methodological approaches can assess the effects of disease-associated SLC19A3 mutations on protein localization?

Investigating the impact of SLC19A3 mutations on protein localization requires systematic approaches:

• Cellular models:

  • Transfection of wild-type vs. mutant SLC19A3 constructs in appropriate cell lines

  • CRISPR/Cas9 knock-in of specific mutations in neuronal or endothelial cell models

  • Patient-derived induced pluripotent stem cells differentiated into relevant cell types

• Analytical techniques:

  • Live-cell imaging with fluorescently tagged SLC19A3 variants

  • FITC-conjugated antibody staining (1:50-1:200) of fixed cells expressing various mutations

  • Subcellular fractionation followed by western blotting (1:500 dilution)

• Mutation panel analysis:

  • Systematic comparison across multiple known disease mutations

  • Classification based on trafficking defects vs. functional defects

  • Correlation with clinical severity and treatment responsiveness

Previous studies have shown that different mutations can either prevent transport of SLC19A3 to the cell surface or reduce the transporter's affinity for thiamine . FITC-conjugated antibodies targeting amino acids 217-246 or 150-300 provide tools to visualize these distinct mechanisms of dysfunction.

How can FITC-conjugated SLC19A3 antibodies facilitate research on therapeutic interventions for thiamine transport disorders?

FITC-conjugated SLC19A3 antibodies enable several research approaches for therapeutic development:

• Pharmacological screening:

  • High-content imaging of SLC19A3 trafficking in response to candidate compounds

  • Quantification of membrane localization as a readout of trafficking rescue

  • Correlation with functional thiamine uptake measurements

• Biotin and thiamine supplementation studies:

  • Dose-response analysis of SLC19A3 expression following treatment

  • Time-course evaluation of protein localization changes

  • Comparative analysis across different mutant variants

• Gene therapy evaluation:

  • Assessment of transgene expression and localization using dual-label approaches

  • Visualization of SLC19A3 restoration in disease models

  • Correlation with functional and clinical endpoints

These approaches address the ongoing research question of why biotin supplementation is effective despite not being a substrate for the thiamine transporter . The hypothesis that biotin regulates SLC19A3 gene transcription can be investigated by combining FITC-conjugated antibody staining with mRNA quantification techniques.

What are the most common technical challenges when using FITC-conjugated SLC19A3 antibodies and their solutions?

Researchers commonly encounter several technical issues when working with FITC-conjugated SLC19A3 antibodies:

For SLC19A3 specifically, its localization to vascular structures means that whole-tissue staining may appear inconsistent if vessels are unevenly distributed. Using vascular markers in parallel can help differentiate between technical issues and true biological variation.

How can researchers optimize fixation and permeabilization protocols for SLC19A3 detection?

Optimizing fixation and permeabilization is critical for successful SLC19A3 antibody staining:

• Fixation considerations:

  • 4% formaldehyde has been validated for SLC19A3 detection

  • 10-20 minute fixation at room temperature is typically sufficient

  • Over-fixation can mask epitopes in the 217-246 or 150-300 amino acid regions

• Permeabilization optimization:

  • 0.2% Triton X-100 has been validated for SLC19A3 antibody access

  • For membrane proteins like SLC19A3, milder detergents (0.1% saponin) may preserve native conformation

  • Duration of permeabilization (5-15 minutes) should be empirically determined

• Antigen retrieval considerations:

  • Heat-induced epitope retrieval may be necessary for some fixed tissues

  • Citrate buffer (pH 6.0) is generally compatible with subsequent FITC detection

  • Enzymatic retrieval methods should be avoided as they may damage the epitope regions

These parameters should be systematically tested when establishing new experimental systems, particularly when working with different tissue types or disease models.

What emerging technologies can enhance SLC19A3 research using immunofluorescence approaches?

Several advanced technologies offer promising applications for SLC19A3 research:

• Super-resolution microscopy:

  • Structured illumination microscopy (SIM) for improved visualization of SLC19A3 distribution in blood vessels

  • Stochastic optical reconstruction microscopy (STORM) for nanoscale localization

  • Expansion microscopy for physical magnification of SLC19A3 distribution patterns

• Multiplexed imaging:

  • Cyclic immunofluorescence for co-detection of SLC19A3 with multiple markers

  • Mass cytometry imaging for highly multiplexed protein detection

  • Spatial transcriptomics combined with SLC19A3 protein detection

• Live imaging approaches:

  • Antibody fragment-based live-cell imaging

  • Correlative light and electron microscopy for ultrastructural context

  • Intravital microscopy for in vivo SLC19A3 dynamics

These advanced approaches could provide unprecedented insights into the polarized distribution of SLC19A3 at the blood-brain barrier and how this distribution is altered in disease states.

How might SLC19A3 antibodies contribute to personalized medicine approaches for thiamine metabolism disorders?

FITC-conjugated SLC19A3 antibodies could support precision medicine initiatives:

• Diagnostic applications:

  • Immunophenotyping of patient-derived cells to classify SLC19A3 mutations

  • Rapid screening for trafficking vs. functional defects

  • Correlation of cellular phenotypes with treatment responsiveness

• Therapeutic monitoring:

  • Assessment of treatment effects on SLC19A3 expression and localization

  • Development of surrogate biomarkers for treatment efficacy

  • Identification of non-responders who might require alternative approaches

• Drug development pipeline:

  • High-throughput screening for compounds that rescue specific mutation types

  • Identification of mutation-specific therapeutic strategies

  • Development of targeted approaches based on mechanistic understanding

The polarized distribution of thiamine transporters in the brain suggests that both SLC19A2 and SLC19A3 are required for transport across the blood-brain barrier . This insight could inform development of combinatorial therapeutic approaches targeting both transport systems.