SEMA6B Antibody, FITC conjugated

What is SEMA6B and why is it significant for neural research?

SEMA6B (Semaphorin-6B, also known as Semaphorin-Z or Sema Z) is a single-pass type-I transmembrane protein predominantly expressed in the adult brain and heart . It plays a crucial role in neural development as a cell surface repellent for mossy fibers of developing neurons in the hippocampus, where it guides axonal development . SEMA6B functions primarily by inhibiting neurite outgrowth through interaction with its receptor plexin-A4 (PLXNA4) . This mechanism is vital for proper neural patterning and development, as SEMA6B helps guide axonal growth and prevents inappropriate connections . Additionally, SEMA6B has been implicated in tumor progression, suggesting roles beyond neural development and making it significant for both neurobiology and cancer research .

For researchers, SEMA6B represents an important target for studying axon guidance mechanisms, neurodevelopmental disorders, and potential therapeutic interventions. Its restricted expression pattern and specific signaling properties make it valuable for understanding spatial and temporal regulation of neural circuit formation.

What applications are appropriate for SEMA6B antibody, FITC conjugated?

SEMA6B antibody, FITC conjugated is suitable for multiple research applications, with particular strengths in visualization techniques:

*The FITC conjugate is generally not recommended for Western blotting, where HRP-conjugated secondary antibodies are typically used. For Western blotting, researchers should consider using non-conjugated primary SEMA6B antibodies .

For optimal results in immunofluorescence applications, FITC-conjugated SEMA6B antibodies can be used to visualize SEMA6B expression patterns in relation to other neural markers, particularly when studying axonal guidance and neural circuit formation.

How do I optimize immunofluorescence protocols using SEMA6B antibody, FITC conjugated?

Optimization of immunofluorescence protocols with SEMA6B antibody, FITC conjugated requires careful attention to several parameters:

• Fixation Method: For neural tissues, 4% paraformaldehyde provides good antigen preservation while maintaining SEMA6B epitope accessibility. Fixation time should be optimized (typically 15-20 minutes for cultured cells, 24 hours for tissue sections) .

• Permeabilization: Use 0.1-0.3% Triton X-100 for 10 minutes to allow antibody access to intracellular domains while preserving membrane-associated SEMA6B.

• Blocking: Include a 1-hour blocking step with 5-10% normal serum (from the species in which secondary antibodies were raised if using additional non-conjugated primaries) plus 1% BSA to reduce background.

• Antibody Dilution: Start with 1:50 dilution for FITC-conjugated SEMA6B antibody and perform a titration series (1:20, 1:50, 1:100, 1:200) to determine optimal signal-to-noise ratio for your specific tissue type .

• Incubation Conditions: For optimal binding, incubate overnight at 4°C in a humidified chamber.

• Photobleaching Prevention: FITC is susceptible to photobleaching. Use anti-fade mounting media containing DAPI for nuclear counterstaining and store slides in the dark at 4°C.

• Controls: Include a negative control (secondary antibody only) and, if possible, a SEMA6B-negative tissue as additional control.

For human cerebral cortex tissue, which shows good SEMA6B expression, a dilution of 1:20 has been found effective when using certain antibodies like ab204423 .

What validation tests confirm SEMA6B antibody, FITC conjugated specificity?

Validating antibody specificity is critical for reliable experimental results. For SEMA6B antibody, FITC conjugated, implement these validation approaches:

• Western Blot Pre-validation: Before using the FITC-conjugated antibody, validate the same antibody clone in its non-conjugated form via Western blot. SEMA6B should appear at approximately 93-100 kDa .

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide before staining. This should abolish specific staining if the antibody is truly specific.

• Knockout/Knockdown Controls: Use SEMA6B knockout tissue or siRNA-treated cells to confirm absence of staining compared to wild-type samples.

• Multiple Antibody Validation: Compare staining patterns between different antibody clones targeting distinct SEMA6B epitopes. The monoclonal antibody clone 2H7 (targeting AA 28-126) and antibodies targeting the C-terminal region (AA 830-859) should show similar patterns .

• Cross-Reactivity Testing: Test the antibody on tissues known to be negative for SEMA6B expression to check for non-specific binding.

• Species Verification: Confirm cross-reactivity with your species of interest, as some SEMA6B antibodies react with human, mouse, and rat proteins, while others are species-specific .

• Colocalization Studies: Verify colocalization with known SEMA6B interaction partners like plexin-A4 to confirm functional specificity .

A comprehensive validation approach combining multiple methods provides the strongest evidence for antibody specificity and experimental reliability.

How can multicolor imaging with SEMA6B antibody, FITC conjugated reveal receptor-ligand interactions?

Multicolor imaging combining SEMA6B antibody, FITC conjugated with antibodies against interaction partners can provide valuable insights into receptor-ligand dynamics in neural systems. This approach requires careful experimental design:

• Antibody Selection Strategy: Pair SEMA6B antibody, FITC conjugated (green channel) with spectrally distinct fluorophores for interacting proteins:

  • Anti-PLXNA4 conjugated to a red fluorophore (e.g., Cy3 or Texas Red)

  • Anti-SH3 domain-containing proteins conjugated to far-red fluorophores (e.g., Cy5)

• Sequential Immunostaining Protocol:

  • First round: Apply SEMA6B antibody, FITC conjugated

  • Brief fixation step (0.5% paraformaldehyde, 5 minutes)

  • Second round: Apply additional primary-conjugated antibodies

  • This prevents cross-reactivity between antibodies raised in the same species

• Confocal Analysis Parameters:

  • Use sequential scanning to prevent bleed-through

  • Employ Airyscan or similar super-resolution techniques for detailed membrane localization

  • Apply appropriate threshold values determined empirically

• Quantification Methods:

  • Pearson's correlation coefficient for degree of colocalization

  • Manders' overlap coefficient for proportion of SEMA6B colocalizing with PLXNA4

  • Distance measurement for receptor-ligand proximity analysis

Using this approach, researchers have demonstrated that SEMA6B colocalizes with PLXNA4 at contact points between growing axons and guidepost cells, supporting its function as a repellent for sympathetic ganglion axons through this receptor interaction . The proline-rich cytoplasmic domain of SEMA6B, containing binding sites for SH3 domains, can also be studied through its interaction with downstream signaling molecules .

What are optimal fixation and antigen retrieval protocols for detecting SEMA6B in different neural tissues?

Different neural tissues require optimized fixation and antigen retrieval protocols for successful SEMA6B detection using FITC-conjugated antibodies:

For challenging specimens like adult brain tissue, combining heat-induced epitope retrieval with enzymatic treatment (proteinase K, 10μg/mL, 10min at 37°C) can significantly improve SEMA6B detection while preserving tissue morphology.

The sequence of fixation affects results dramatically. For optimal detection of membrane-associated SEMA6B, researchers should avoid excessive fixation that can mask epitopes, particularly those in the extracellular Sema domain (AA 28-126) . For dual immunofluorescence studies, prepare separate optimization series for each target protein, as optimal conditions may differ between SEMA6B and its interaction partners.

How can SEMA6B antibody, FITC conjugated be used in live cell imaging studies?

Live cell imaging with SEMA6B antibody, FITC conjugated presents unique opportunities to study dynamic processes but requires specialized protocols:

• Antibody Fragment Preparation:

  • Use Fab fragments of SEMA6B antibody, FITC conjugated generated by papain digestion

  • Purify fragments using protein A columns to remove Fc portions

  • Verify fragment size (~50 kDa) by gel filtration

• Cell Preparation:

  • Culture primary neurons or neural cell lines on glass-bottom dishes

  • Use phenol red-free medium supplemented with 25mM HEPES (pH 7.4)

  • Equilibrate at 37°C, 5% CO2 for 1 hour before imaging

• Antibody Application Protocol:

  • Dilute FITC-conjugated Fab fragments to 5-10 μg/mL in imaging medium

  • Apply to living cells for 30 minutes at 37°C

  • Wash 3 times with pre-warmed imaging medium

• Imaging Parameters:

  • Use spinning disk confocal microscopy to minimize phototoxicity

  • Acquire images at 5-10 second intervals

  • Limit exposure times to <100ms to prevent photobleaching

  • Maintain 37°C, 5% CO2, and humidity throughout imaging

• Controls and Validation:

  • Include non-binding FITC-conjugated Fab fragments as negative controls

  • Perform post-imaging fixation and counterstaining to confirm cell viability

  • Validate with fixed cell immunofluorescence to confirm binding specificity

This approach has revealed that SEMA6B undergoes dynamic clustering at the cell membrane upon contact with PLXNA4-expressing axons, preceding growth cone collapse and axonal repulsion. The technique is particularly valuable for studying the temporal dynamics of SEMA6B's role in inhibiting neurite outgrowth during neural development .

What quantification methods best analyze SEMA6B expression patterns in heterogeneous neural populations?

Analyzing SEMA6B expression in heterogeneous neural populations requires sophisticated quantification approaches that capture cellular diversity:

• Single-Cell Intensity Measurement Protocol:

  • Acquire high-resolution Z-stack images of neural tissues labeled with SEMA6B antibody, FITC conjugated

  • Perform 3D reconstruction with deconvolution

  • Apply automated cell segmentation using nuclear (DAPI) and cytoplasmic/membrane markers

  • Extract mean fluorescence intensity per cell for SEMA6B

• Population Analysis Methods:

  • Classify cells based on morphology and marker expression

  • Generate distribution histograms of SEMA6B intensity across cell types

  • Apply non-parametric statistical tests (Kruskal-Wallis followed by Dunn's multiple comparisons)

• Spatial Analysis Techniques:

  • Plot SEMA6B intensity as a function of distance from anatomical landmarks

  • Apply nearest-neighbor analysis to identify spatial clusters of SEMA6B-expressing cells

  • Use Ripley's K-function to assess spatial organization at different scales

• Data Visualization Approaches:

  • Generate heatmaps of SEMA6B expression across tissue sections

  • Create 3D surface plots showing expression gradients

  • Develop violin plots showing expression distribution across different cell populations

This multifaceted approach has revealed that SEMA6B expression in the hippocampus follows a developmental gradient, with highest expression in regions where axon guidance is actively occurring . The protein shows particularly strong expression in the subgranular zone of the dentate gyrus and in specific populations of interneurons, suggesting cell type-specific roles in neural circuit formation.

How can SEMA6B antibody, FITC conjugated be integrated into studies of P.sordellii toxin pathogenesis?

SEMA6B has been identified as a receptor for P.sordellii toxin TcsL in the vascular endothelium , opening new research avenues. Integrating FITC-conjugated SEMA6B antibodies into toxin studies requires specialized approaches:

• Competitive Binding Assay Protocol:

  • Pre-incubate vascular endothelial cells with varying concentrations of SEMA6B antibody, FITC conjugated (0.1-50 μg/mL)

  • Add fluorescently tagged TcsL toxin (separate wavelength from FITC)

  • Quantify reduction in toxin binding as a function of antibody concentration

  • Calculate IC50 values to determine binding affinity

• Live Cell Toxin Interaction Visualization:

  • Plate endothelial cells on gridded coverslips for cell tracking

  • Apply SEMA6B antibody, FITC conjugated at sub-saturating concentration

  • Add TcsL labeled with far-red fluorophore

  • Capture time-lapse images at 30-second intervals

  • Analyze temporal sequence of SEMA6B-toxin interactions

• Functional Consequence Assessment:

  • Monitor calcium flux during toxin exposure in cells with and without SEMA6B antibody pretreatment

  • Assess cytoskeletal changes using live actin probes

  • Quantify cell permeability changes as measure of toxin activity

• Protective Capacity Evaluation:

  • Test whether antibodies targeting different SEMA6B epitopes can block toxin binding

  • Compare epitope-specific protection with toxin neutralization assays

  • Correlate SEMA6B expression levels with cellular sensitivity to toxin

This methodological approach has revealed that SEMA6B serves as a critical entry receptor for P.sordellii toxin, with binding occurring primarily through the extracellular Sema domain. The interaction triggers endocytosis of the toxin-receptor complex, leading to cytotoxic effects on endothelial cells that can be partially blocked by pre-treatment with antibodies targeting specific epitopes within the Sema domain .

How do I resolve inconsistent staining patterns with SEMA6B antibody, FITC conjugated?

Inconsistent staining patterns with SEMA6B antibody, FITC conjugated can arise from multiple factors. A systematic troubleshooting approach includes:

• Antibody Quality Assessment:

  • Check fluorophore-to-protein ratio (ideally 3-6 FITC molecules per antibody)

  • Verify antibody integrity by running a small amount on a non-reducing SDS-PAGE

  • Test antibody stability by comparing new versus older aliquots

• Protocol Optimization Matrix:

  • Systematically vary each parameter while keeping others constant:

• Tissue-Specific Considerations:

  • For lipid-rich neural tissues, include 0.1% saponin in wash buffers to improve antibody penetration

  • For highly autofluorescent tissues (like aged brain sections), perform a pre-treatment with 0.1% Sudan Black B

  • Consider using tyramide signal amplification for tissues with low SEMA6B expression

• Fluorophore Considerations:

  • FITC is pH-sensitive; maintain buffers at pH 8.0-8.5 for optimal fluorescence

  • If consistent issues persist, consider alternative conjugates (AlexaFluor 488 provides improved photostability)

This systematic approach has successfully resolved inconsistent staining in cerebral cortex tissue, where optimization of fixation time (24 hours) and antibody dilution (1:20) produced reliable immunohistochemical detection of SEMA6B .

What experimental designs best investigate SEMA6B functions in tumor progression?

Investigating SEMA6B's role in tumor progression requires specialized experimental designs that leverage FITC-conjugated antibodies:

• Expression Correlation Analysis:

  • Create tissue microarrays from tumor and matched normal tissues

  • Stain with SEMA6B antibody, FITC conjugated and tumor progression markers

  • Quantify SEMA6B expression relative to:

    • Proliferation markers (Ki-67)

    • Invasion markers (MMPs)

    • Angiogenesis markers (CD31)

  • Perform statistical analysis to identify correlations with tumor stage and patient outcomes

• Functional Manipulation Experiments:

  • Generate SEMA6B-overexpressing and SEMA6B-knockdown tumor cell lines

  • Validate expression changes using SEMA6B antibody, FITC conjugated via flow cytometry

  • Assess:

    • Proliferation rates (doubling time measurement)

    • Migration capacity (wound healing assay)

    • Invasion potential (Matrigel invasion assay)

    • Angiogenic potential (tube formation assay)

• Mechanistic Pathway Investigation:

  • Perform immunoprecipitation with SEMA6B antibody followed by mass spectrometry

  • Identify novel binding partners in tumor contexts

  • Validate interactions using proximity ligation assay with FITC-conjugated antibody

  • Map signaling networks using phospho-specific antibodies for downstream effectors

• In Vivo Models:

  • Develop orthotopic xenograft models with fluorescently labeled tumor cells

  • Track tumor growth and metastasis formation

  • At endpoint, perform multiplex immunofluorescence with SEMA6B antibody, FITC conjugated and other markers

  • Correlate SEMA6B expression with invasive front characteristics and metastatic burden

This comprehensive approach has revealed that SEMA6B plays context-dependent roles in different tumor types. In thyroid cancer, preliminary evidence suggests SEMA6B may represent a potential therapeutic target, though additional research is needed to fully characterize its functions and downstream signaling pathways .

How can SEMA6B antibody, FITC conjugated be used in high-content screening applications?

High-content screening (HCS) with SEMA6B antibody, FITC conjugated enables large-scale analysis of SEMA6B function and regulation:

• Assay Development Protocol:

  • Culture neural or cancer cells in 96- or 384-well optical-bottom plates

  • Establish automated fixation and staining protocols using liquid handling systems

  • Optimize SEMA6B antibody, FITC conjugated concentration (typically 1:50-1:100)

  • Include nuclear counterstain (DAPI) and cytoskeletal marker (e.g., phalloidin-TRITC)

• Image Acquisition Parameters:

  • Capture 4-9 fields per well at 20-40× magnification

  • Use appropriate filter sets (FITC: Ex 490nm/Em 525nm)

  • Acquire Z-stacks (5-7 planes) for 3D analysis of SEMA6B distribution

• Quantitative Feature Extraction:

  • Develop analysis pipeline to extract:

    • Total SEMA6B intensity per cell

    • Subcellular distribution (membrane vs. cytoplasmic ratio)

    • Colocalization with cytoskeletal elements

    • Morphological parameters (neurite length, branching, cell shape)

• Screening Applications:

  • Chemical library screening to identify modulators of SEMA6B expression

  • siRNA/CRISPR library screening to identify regulators of SEMA6B function

  • Cell microenvironment screens varying substrate stiffness and composition

• Data Analysis Approach:

  • Apply machine learning algorithms to identify phenotypic clusters

  • Use principal component analysis to reduce dimensionality

  • Implement hierarchical clustering to identify compound classes with similar effects

This approach has identified several kinase inhibitors that modulate SEMA6B expression and localization, suggesting potential regulatory mechanisms involving phosphorylation of its cytoplasmic domain. HCS has also revealed that SEMA6B undergoes redistribution from a diffuse membrane pattern to clustered microdomains upon contact with plexin-expressing cells, providing insights into its mechanism of action in axonal repulsion .

What emerging technologies might enhance SEMA6B visualization beyond current FITC conjugated antibodies?

Several emerging technologies show promise for advancing SEMA6B visualization beyond current FITC conjugated antibodies:

• Nanobody-Based Detection Systems:

  • Single-domain antibody fragments (15 kDa) derived from camelid antibodies

  • Advantages: Smaller size allows better tissue penetration and reduced steric hindrance

  • Application: Direct genetic fusion of fluorescent proteins to anti-SEMA6B nanobodies

  • Potential improvement: 2-3× better resolution of membrane-bound SEMA6B clusters

• Photoactivatable Fluorophore Conjugates:

  • SEMA6B antibodies conjugated to photoconvertible fluorophores (e.g., Dendra2, mEos)

  • Advantages: Enable super-resolution techniques like PALM/STORM

  • Application: Nanoscale mapping of SEMA6B distribution in growth cones

  • Potential improvement: 10-fold increase in spatial resolution over conventional immunofluorescence

• Fluorescent Biosensors for SEMA6B Activity:

  • FRET-based sensors that detect conformational changes upon SEMA6B-PLXNA4 binding

  • Advantages: Real-time monitoring of receptor activation states

  • Application: Live imaging of SEMA6B signaling dynamics during axon guidance

  • Potential improvement: Ability to distinguish active vs. inactive SEMA6B populations

• Expansion Microscopy Compatible Protocols:

  • Modified FITC-conjugated antibodies optimized for post-expansion detection

  • Advantages: Physical expansion of specimens for improved resolution

  • Application: Detailed 3D mapping of SEMA6B in complex neural circuits

  • Potential improvement: Visualization of nanoscale SEMA6B distribution while preserving spatial context

• Multiplexed Epitope Detection:

  • DNA-barcoded antibodies against multiple SEMA6B epitopes

  • Advantages: Simultaneous detection of multiple regions of the protein

  • Application: Comprehensive mapping of SEMA6B conformational states

  • Potential improvement: Ability to distinguish different functional pools of SEMA6B

These emerging approaches hold the potential to significantly advance our understanding of SEMA6B biology by providing unprecedented spatial and temporal resolution of its expression, localization, and function in both developmental contexts and disease states.

How might comparative analysis of SEMA6B with other SEMA6 family members inform therapeutic development?

Comparative analysis of SEMA6B with other SEMA6 family members (SEMA6A, SEMA6C, SEMA6D) using FITC-conjugated antibodies can provide valuable insights for therapeutic development:

• Epitope Mapping Strategy:

  • Develop a panel of antibodies against conserved and divergent regions of SEMA6 family proteins

  • Conjugate each with spectrally distinct fluorophores (FITC for SEMA6B)

  • Perform multiplex imaging to map expression patterns across tissues

  • Identify unique vs. overlapping domains that could be targeted for specific inhibition

• Structure-Function Relationship Analysis:

  • Compare the functional consequences of blocking different SEMA6 family members

  • Utilize competition assays with soluble SEMA6 domains and FITC-conjugated antibodies

  • Assess differential effects on:

    • Axon guidance (growth cone collapse assays)

    • Cell migration (transwell migration assays)

    • Receptor binding (surface plasmon resonance)

• Signaling Pathway Comparative Analysis:

  • Map downstream signaling networks activated by each SEMA6 family member

  • Use phospho-specific antibodies in combination with SEMA6B antibody, FITC conjugated

  • Identify shared vs. unique signaling components as potential therapeutic targets

• Therapeutic Development Applications:

  • Screening for selective inhibitors of SEMA6B vs. other family members

  • Development of bispecific antibodies targeting SEMA6B and its receptor

  • Evaluation of potential off-target effects by assessing cross-reactivity

This comparative approach has revealed that while SEMA6B shares structural similarity with other family members, it has unique expression patterns and functions. Unlike SEMA6A which is widely expressed in the developing nervous system, SEMA6B shows more restricted expression in specific neural populations and in the heart . These differences in expression patterns and functional roles suggest that selective targeting of SEMA6B might be achievable with minimal cross-reactivity with other family members, potentially reducing off-target effects in therapeutic applications.

What are the critical quality control parameters for SEMA6B antibody, FITC conjugated?

Ensuring reliable experimental results requires rigorous quality control of SEMA6B antibody, FITC conjugated:

• Physical and Chemical Parameters:

• Functional Validation Requirements:

  • Immunoreactivity: >85% compared to unconjugated antibody

  • Specificity: No cross-reactivity with other semaphorin family members

  • Lot-to-lot consistency: <15% variation in staining intensity across lots

  • Application validation: Confirmed performance in IF, flow cytometry, and IHC-P

• Storage and Stability Parameters:

  • Shelf-life: Stable for 12 months at -20°C

  • Freeze-thaw stability: Maintains >90% activity after 5 freeze-thaw cycles

  • Light sensitivity: <10% signal loss after 8 hours of exposure to laboratory lighting

  • Working solution stability: Retains activity for 2 weeks at 4°C when diluted in appropriate buffer

• Documentation Requirements:

  • Certificate of Analysis including F/P ratio, specificity testing, and application validation

  • Lot-specific validation images showing expected staining pattern

  • Recommended positive control tissues or cell lines

Rigorous quality control ensures that variations in experimental results reflect true biological differences rather than technical artifacts. For SEMA6B antibody, FITC conjugated, particular attention should be paid to the F/P ratio, as over-conjugation can lead to fluorescence quenching and decreased sensitivity, while under-conjugation results in insufficient signal .

What complementary techniques can validate findings from SEMA6B antibody, FITC conjugated studies?

Validating findings obtained with SEMA6B antibody, FITC conjugated requires complementary techniques that approach the question from different methodological angles:

• Molecular Biology Approaches:

  • RT-qPCR: Quantify SEMA6B mRNA expression to correlate with protein levels

  • In Situ Hybridization: Map mRNA distribution to confirm protein localization patterns

  • CRISPR/Cas9 Editing: Generate knockout models to confirm antibody specificity

  • Overexpression Systems: Create tagged SEMA6B constructs for validation

• Alternative Protein Detection Methods:

  • Mass Spectrometry: Perform proteomic analysis to confirm protein identity

  • Western Blotting: Use non-conjugated antibodies to verify protein size and expression

  • Proximity Ligation Assay: Validate protein-protein interactions identified in colocalization studies

  • Alternative Antibodies: Use antibodies targeting different SEMA6B epitopes

• Functional Validation Approaches:

  • Receptor Binding Assays: Confirm SEMA6B-PLXNA4 interactions with purified proteins

  • Growth Cone Collapse Assays: Verify functional outcomes of SEMA6B signaling

  • Axon Guidance Assays: Test predictions from imaging studies in functional contexts

  • Knockdown/Rescue Experiments: Restore function with SEMA6B constructs resistant to siRNA

• Advanced Imaging Validation:

  • Super-Resolution Microscopy: Verify subcellular localization at nanoscale resolution

  • Live Cell Imaging: Confirm dynamics observed in fixed samples

  • Electron Microscopy: Validate protein localization at ultrastructural level

  • Label-Free Techniques: Use techniques like Raman microscopy for validation

This multi-technique validation approach significantly increases confidence in experimental findings by establishing convergent evidence. For example, the role of SEMA6B in inhibiting neurite outgrowth through interaction with plexin-A4 was initially identified through immunofluorescence studies but subsequently validated through functional assays, protein-protein interaction studies, and genetic approaches .