TNR Antibody, FITC conjugated

What is Tenascin R (TNR) and what is its biological significance?

Tenascin R (TNR), also known as Restrictin or Janusin, is a neural extracellular matrix (ECM) protein that belongs to the tenascin family. It plays crucial roles in neural tissue through interactions with different cells and matrix components. These interactions can significantly influence cellular behavior by either promoting stable adhesion and differentiation or causing repulsion and inhibition of neurite growth. TNR functions by binding to cell surface gangliosides, which can inhibit RGD-dependent integrin-mediated cell adhesion and result in inhibition of PTK2 (FAK) phosphorylation . The protein has a calculated molecular weight of approximately 150 kDa but is typically observed at 160-180 kDa in experimental contexts, likely due to post-translational modifications .

What are FITC-conjugated TNR antibodies and their primary applications?

FITC-conjugated TNR antibodies are immunological reagents where fluorescein isothiocyanate (FITC) has been chemically linked to antibodies that specifically recognize Tenascin R. This fluorescent conjugation enables direct visualization of TNR in various experimental techniques. These antibodies are primarily used for immunofluorescent staining in applications such as flow cytometry, immunohistochemistry (IHC), and immunocytochemistry (ICC). They allow researchers to identify and enumerate TNR-expressing cells within mixed cell populations . Based on available reagents, FITC-conjugated anti-TNR antibodies targeting the amino acid sequence 1231-1319 of the human TNR protein are commercially available .

What reactivity patterns can be expected with TNR antibodies across species?

TNR antibodies exhibit various cross-reactivity profiles depending on their specific epitope targets. Based on the available data, certain TNR antibodies demonstrate reactivity across multiple species:

• Human-reactive TNR antibodies are widely available, with applications in ELISA, IHC, and western blotting

• Rodent-reactive antibodies (mouse and rat) are common, particularly for neural tissue studies

• Some antibodies show broader cross-reactivity profiles, including chicken and cow samples

When selecting a TNR antibody for research, it's essential to verify the specific reactivity profile for your species of interest, as epitope conservation varies across evolutionary lineages.

What are the optimal experimental conditions for using FITC-conjugated TNR antibodies in multicolor flow cytometry?

For multicolor flow cytometric analyses using FITC-conjugated TNR antibodies, several critical parameters must be optimized:

• Antibody Titration: FITC-conjugated antibodies should be carefully titrated, typically using ≤0.5 μg mAb per million cells to determine the optimal concentration that maximizes signal-to-noise ratio . Titration curves are essential to avoid both inadequate staining and excessive background.

• Compensation Controls: When used in multicolor panels, single-stained controls with the FITC-conjugated antibody are necessary for accurate compensation, as FITC has significant spectral overlap with other fluorochromes like PE.

• Instrument Settings: Proper instrument settings for FITC detection require excitation at 488 nm and emission detection around 525 nm. Voltage adjustments should be optimized to place positive populations within the detector's linear range .

• Controls: Incorporating appropriate controls is crucial, including:

  • Isotype controls (e.g., FITC-MOPC-21) at comparable concentrations to assess background staining

  • Blocking controls, either by pre-blocking the FITC-conjugated antibody with excess recombinant TNR or pre-blocking cells with unlabeled TNR antibody

• Cell Preparation: For intracellular TNR detection, optimal fixation and permeabilization methods typically involve paraformaldehyde fixation followed by saponin permeabilization .

How can TNR antibodies contribute to neural extracellular matrix research?

TNR antibodies serve as powerful tools for investigating the neural extracellular matrix because of TNR's specialized functions in neural development and maintenance. Research applications include:

• Cell-Matrix Interaction Studies: TNR antibodies can help visualize and quantify TNR's interactions with cellular components and other matrix molecules, providing insight into mechanisms of neuronal adhesion, repulsion, and guidance .

• Neural Development Research: By tracking TNR expression patterns during development, researchers can investigate how this protein influences the formation of neural circuits and structures.

• Pathological Investigations: TNR antibodies allow researchers to examine alterations in TNR expression or localization in neurological disorders or injury models, potentially revealing mechanisms of neural dysfunction.

• Functional Studies: TNR antibodies can be used in neutralization experiments to block TNR function, helping to elucidate its role in processes such as neurite outgrowth inhibition and synaptic plasticity.

• Receptor Identification: Through co-immunoprecipitation or proximity ligation assays, TNR antibodies can help identify receptors or binding partners that mediate TNR's effects on neural cells.

What methodological considerations are important when studying TNR expression in human brain tissue using immunohistochemistry?

When conducting immunohistochemical analysis of TNR in human brain tissue, several technical considerations are essential:

• Antigen Retrieval: For optimal TNR detection in formalin-fixed, paraffin-embedded human brain tissue, antigen retrieval with TE buffer at pH 9.0 is recommended, though citrate buffer at pH 6.0 may be used as an alternative .

• Antibody Dilution: Appropriate antibody dilution ranges for TNR immunohistochemistry typically fall between 1:20 and 1:200, requiring optimization for specific tissue preparation methods and antibody lots .

• Controls and Validation: Positive controls should include human brain tissue known to express TNR, while negative controls should involve either antibody omission or isotype-matched control antibodies. Multiple antibodies targeting different TNR epitopes can provide validation of staining patterns.

• Signal Amplification: When studying TNR in tissues with lower expression levels, signal amplification systems such as tyramide signal amplification may be necessary.

• Co-localization Studies: Combining TNR immunostaining with markers for specific cell types or subcellular structures can provide valuable functional insights. For FITC-conjugated antibodies, consideration of autofluorescence quenching methods may be necessary, particularly in aged human brain tissue.

What is the recommended protocol for immunofluorescence staining using FITC-conjugated TNR antibodies?

A standardized protocol for immunofluorescence staining with FITC-conjugated TNR antibodies includes:

• Sample Preparation:

  • For cell cultures: Fix cells with 4% paraformaldehyde for 15 minutes at room temperature

  • For tissue sections: Use freshly frozen or properly fixed and processed tissue sections (4-10 μm thickness)

• Blocking and Permeabilization:

  • Permeabilize with 0.1-0.3% Triton X-100 in PBS for 10 minutes

  • Block with 5-10% normal serum (from the species of the secondary antibody) and 1% BSA in PBS for 1 hour

• Primary Antibody Incubation:

  • Dilute FITC-conjugated TNR antibody (typically 1:500 for ICC applications)

  • Incubate overnight at 4°C or 1-2 hours at room temperature in a humidified chamber

• Washing:

  • Wash 3-5 times with PBS, 5 minutes each wash

• Counterstaining:

  • Counterstain nuclei with DAPI (1 μg/ml) for 5 minutes

• Mounting:

  • Mount with anti-fade mounting medium to minimize photobleaching

• Imaging:

  • Use appropriate filter sets for FITC (excitation ~495 nm, emission ~520 nm)

  • Minimize exposure time to reduce photobleaching

What controls should be included when working with FITC-conjugated TNR antibodies?

A comprehensive control strategy for experiments using FITC-conjugated TNR antibodies should include:

• Isotype Controls:

  • Include FITC-conjugated isotype-matched control antibodies (e.g., FITC-MOPC-21 for mouse IgG1 antibodies) to assess background staining levels

  • Use at the same concentration as the primary antibody

• Blocking Controls:

  • Pre-block the FITC-conjugated antibody with molar excess of recombinant TNR protein

  • Alternatively, pre-block the fixed/permeabilized cells with unlabeled TNR antibody prior to staining with the FITC-conjugated version

• Autofluorescence Controls:

  • Include unstained samples to assess natural tissue autofluorescence

  • Consider using autofluorescence quenching reagents when working with tissues known to have high autofluorescence (e.g., brain tissue)

• Positive Controls:

  • Include samples known to express TNR, such as human brain tissue or SH-SY5Y cells

• Negative Controls:

  • Include samples known not to express TNR

  • Omit primary antibody while maintaining all other steps of the protocol

How can researchers optimize western blotting protocols for TNR detection?

Optimizing western blotting for TNR detection requires several specific considerations:

• Sample Preparation:

  • Human brain tissue or TNR-expressing cell lines like SH-SY5Y are appropriate positive controls

  • Use protein extraction buffers containing protease inhibitors to prevent degradation

  • Due to TNR's high molecular weight (observed at 160-180 kDa), use lower percentage gels (6-8% acrylamide) for better resolution

• Gel Electrophoresis and Transfer:

  • For large proteins like TNR, extended transfer times or specialized transfer conditions may be necessary

  • Consider semi-dry or wet transfer systems with modified buffers for high molecular weight proteins

• Antibody Dilution and Incubation:

  • For western blotting, use TNR antibodies at dilutions between 1:500 and 1:2000

  • Extend primary antibody incubation to overnight at 4°C for optimal binding

• Detection System:

  • For FITC-conjugated TNR antibodies in western blotting, fluorescence imaging systems are required

  • For non-conjugated antibodies, standard HRP-conjugated secondary antibodies with chemiluminescent detection work well

• Expected Results:

  • TNR typically appears as bands at approximately 160-180 kDa

  • Multiple bands may be observed due to alternative splicing or post-translational modifications

What are the best approaches for troubleshooting weak signals when using FITC-conjugated TNR antibodies?

When faced with weak signals using FITC-conjugated TNR antibodies, researchers should consider these troubleshooting approaches:

• Antibody Concentration Optimization:

  • Increase antibody concentration incrementally, testing a range above the recommended dilution

  • For TNR antibodies in immunohistochemistry, the recommended range is 1:20-1:200; consider using the higher concentration end for weak signals

• Sample Preparation Improvements:

  • Optimize fixation conditions - overfixation can mask epitopes while underfixation may compromise tissue morphology

  • Test alternative antigen retrieval methods (TE buffer pH 9.0 is recommended for TNR, but citrate buffer pH 6.0 can be tried as an alternative)

• Signal Amplification Methods:

  • Consider tyramide signal amplification (TSA) for immunohistochemistry

  • For flow cytometry, examine alternative fluorophores with higher quantum yields than FITC

• Reducing Background Interference:

  • Use proper blocking reagents (5-10% serum plus 1% BSA)

  • Include detergents in wash buffers to reduce non-specific binding

  • For tissues with high autofluorescence, consider quenching treatments or switching to non-fluorescent detection methods

• Storage and Handling:

  • FITC is sensitive to photobleaching; store conjugated antibodies in the dark

  • Aliquot antibodies to avoid repeated freeze-thaw cycles

  • For long-term storage, keep at -20°C in buffer containing 50% glycerol and 0.02% sodium azide

How can FITC-conjugated TNR antibodies be effectively used in multicolor immunofluorescence studies?

To effectively incorporate FITC-conjugated TNR antibodies in multicolor immunofluorescence:

• Spectral Considerations:

  • Plan fluorophore combinations carefully to minimize spectral overlap

  • FITC (excitation ~495 nm, emission ~520 nm) has significant overlap with other green fluorophores

• Panel Design:

  • Pair FITC with fluorophores in distinctly different channels (e.g., DAPI for nuclei, Cy5 or Alexa 647 for other markers)

  • When studying TNR in relation to other neural markers, consider this sample panel design:

    • FITC-conjugated TNR antibody

    • Alexa 647-conjugated antibody for neuronal markers

    • DAPI for nuclear counterstain

• Sequential Staining Approach:

  • For complex tissues or when antibody species conflicts exist, use sequential staining protocols

  • Apply FITC-conjugated antibodies later in the sequence to minimize exposure to washing steps

• Imaging Optimization:

  • Capture FITC channel early in the imaging sequence to minimize photobleaching

  • Use appropriate exposure settings to balance signal intensity against photobleaching

• Analysis Considerations:

  • Apply spectral unmixing algorithms if significant bleed-through occurs

  • Establish careful thresholding based on control samples

A proposed staining panel for neural tissue based on published methods would include:

How do the applications of TNR antibodies differ between flow cytometry and microscopy techniques?

TNR antibodies serve distinct purposes across different experimental platforms:

In Flow Cytometry:

• FITC-conjugated TNR antibodies allow quantitative analysis of TNR expression levels across cell populations

• For optimal flow cytometric analysis, FITC-conjugated antibodies should be carefully titrated (≤0.5 μg per million cells)

• Flow cytometric applications are particularly useful for examining TNR expression in neural precursor cells or in experimental models of neural development

• Cell preparation typically involves fixation and permeabilization protocols, as TNR may have both surface and intracellular epitopes

In Microscopy:

• Immunohistochemistry with TNR antibodies reveals the spatial distribution of TNR within tissue architecture, essential for understanding its role in the neural extracellular matrix

• For immunohistochemistry, recommended dilutions range from 1:20 to 1:200, depending on the specific antibody and tissue preparation

• Immunocytochemistry applications (typically at 1:500 dilution) allow visualization of TNR in cultured cells, enabling studies of its cellular localization and trafficking

• Microscopy techniques can be combined with other markers to examine TNR's relationships with cellular structures and other extracellular matrix components

What considerations are important when selecting between different TNR antibody epitopes for research?

Selecting the appropriate TNR antibody epitope is critical for experimental success:

• Functional Domains:

  • TNR contains multiple functional domains with distinct biological activities

  • Antibodies targeting amino acids 329-588 may be suitable for studying TNR's cell adhesion functions

  • Antibodies against the C-terminal region (AA 1231-1319) are commercially available for human samples

• Species Conservation:

  • Epitope conservation varies across species; some TNR regions show higher conservation between humans and rodents

  • For cross-species studies, selecting antibodies against highly conserved regions is advantageous

  • Available antibodies demonstrate reactivity across human, rat, mouse, chicken, and cow samples

• Accessibility in Native Protein:

  • Consider whether the epitope is accessible in the protein's native conformation

  • N-terminal antibodies (e.g., those targeting AA 104-117) may be useful for surface expression studies

  • C-terminal antibodies might be better for detecting processed forms of the protein

• Application Compatibility:

  • Some epitopes perform better in specific applications:

    • The AA 1059-1358 region has been validated for western blotting and immunofluorescence

    • The AA 411-519 epitope has been validated for ELISA and western blotting

When designing experiments, researchers should select epitopes that align with their specific research questions and technical requirements.

How should researchers design experiments to study TNR's role in neural development and pathology?

Effective experimental design for TNR studies should consider:

• Model Selection:

  • In vitro models: Primary neuronal cultures, neural stem cells, or established neural cell lines (such as SH-SY5Y that express TNR)

  • In vivo models: Transgenic models with TNR modifications or species where TNR antibodies show validated reactivity (human, mouse, rat)

• Temporal Considerations:

  • TNR expression varies during development and in response to neural injury or disease

  • Time-course studies are essential to capture dynamic changes

• Functional Approaches:

  • Loss-of-function studies using TNR knockdown or knockout models

  • Gain-of-function approaches through TNR overexpression

  • Blocking studies using anti-TNR antibodies to neutralize specific functions

• Analytical Methods:

  • Combine multiple techniques (immunohistochemistry, western blotting, and functional assays)

  • Quantitative analysis of TNR expression using image analysis software for immunohistochemistry or flow cytometry

  • Co-localization studies with other neural markers to establish cellular context

• Validation Strategies:

  • Use multiple antibodies targeting different TNR epitopes

  • Incorporate molecular techniques (RT-PCR, RNA sequencing) to confirm expression patterns

What data analysis approaches are recommended for quantifying TNR expression in immunofluorescence studies?

For rigorous quantification of TNR expression using immunofluorescence:

• Image Acquisition Standards:

  • Consistent exposure settings across experimental groups

  • Multiple representative fields per sample (minimum 5-10)

  • Z-stack imaging for tissue sections to capture the full signal distribution

• Preprocessing Steps:

  • Background subtraction based on negative control samples

  • Thresholding to distinguish specific signal from background

  • For FITC-labeled samples, autofluorescence correction may be necessary

• Quantification Metrics:

  • Mean fluorescence intensity within regions of interest

  • Area of positive staining as a percentage of total tissue area

  • Co-localization coefficients (Pearson's or Mander's) when examining TNR in relation to other markers

• Statistical Analysis:

  • Normality testing before selecting parametric or non-parametric tests

  • Use of appropriate statistical tests based on experimental design (t-tests, ANOVA, or non-parametric alternatives)

  • Report both effect sizes and p-values for comprehensive interpretation

• Visualization Methods:

  • Box plots or violin plots for distribution data

  • Representative images alongside quantitative results

  • Heat maps for spatial distribution analysis

Using these approaches ensures reproducible and statistically sound quantification of TNR expression patterns in complex neural tissues.