TNN Antibody, FITC conjugated

What is TNN Antibody, FITC conjugated and what are its primary applications?

TNN Antibody, FITC conjugated is a polyclonal antibody that reacts specifically with human Tenascin-N protein. Tenascin-N (also known as Tenascin-W or TN-W) is an extracellular matrix protein with UniprotID Q9UQP3. The antibody is conjugated to fluorescein isothiocyanate (FITC), a fluorochrome with excitation and emission wavelengths at approximately 495nm and 525nm, providing visible yellow-green fluorescence when excited with ultraviolet or blue light .

Primary applications include:

• Immunofluorescent staining for microscopy

• Flow cytometric analysis

• ELISA-based detection systems

• Identification of TNN-expressing cells in mixed populations

• Tissue section analysis in histopathology studies

The specific reactivity against human samples makes this antibody particularly valuable for studies focusing on human tissue or cell line experiments .

How does FITC conjugation affect antibody functionality?

FITC conjugation to proteins, including antibodies, is a relatively straightforward process that generally does not significantly alter the biological activity or binding specificity of the labeled protein. The conjugation typically occurs via the APTES molecule, forming a covalent bond that is confirmed by the disappearance of the characteristic -N=C=S group peak at approximately 2031 cm⁻¹ in FTIR spectroscopy .

When FITC is conjugated to the TNN antibody:

• Binding specificity to the target antigen (Tenascin-N) remains intact

• The antibody maintains its recognition of the epitope within the recombinant Human Tenascin-N protein (441-556AA)

• Spectral properties shift slightly, with FITC-antibody conjugates typically showing peak absorption at around 494nm (compared to 498nm for FITC-APTES conjugates in ethanol)

The primary advantage of this conjugation is the direct visualization of target binding without requiring secondary detection reagents, streamlining experimental workflows .

What are the optimal storage conditions for TNN Antibody, FITC conjugated?

Proper storage of TNN Antibody, FITC conjugated is critical for maintaining its functionality and fluorescence intensity. The recommended storage conditions include:

• Store at -20°C or -80°C upon receipt

• Avoid repeated freeze-thaw cycles, which can degrade both antibody binding capacity and fluorescence intensity

• Store in the buffer supplied (typically containing 50% Glycerol, 0.01M PBS, pH 7.4, with 0.03% Proclin 300 as preservative)

• Protect from light to prevent photobleaching of the FITC fluorophore

For working solutions, storage at 4°C is acceptable for short periods (1-2 weeks), but long-term storage should follow the freezer conditions mentioned above. Aliquoting the antibody upon receipt can help prevent degradation from repeated freeze-thaw cycles .

What is the difference between TNN and TNF antibodies with FITC conjugation?

While both are FITC-conjugated antibodies, they target entirely different proteins and have distinct research applications:

These differences highlight the importance of selecting the appropriate antibody conjugate based on the specific research target and experimental design .

What controls should be implemented when using TNN Antibody, FITC conjugated for flow cytometry?

When designing flow cytometry experiments with TNN Antibody, FITC conjugated, several controls should be implemented to ensure data reliability:

• Isotype control: Use rabbit IgG-FITC at the same concentration as the TNN antibody to assess background binding and establish gating parameters.

• Blocking control: Pre-block with unlabeled TNN antibody or recombinant TNN protein to demonstrate binding specificity. The disappearance of signal in blocked samples confirms specificity.

• Unstained cells: Essential for establishing autofluorescence levels and determining proper compensation settings.

• Single-color controls: If performing multicolor flow cytometry, single-stained samples are necessary for compensation setup.

• Known positive and negative controls: Cell lines or samples with confirmed TNN expression profiles help validate staining patterns.

For optimal results, titration of the antibody should be performed (similar to the ≤ 0.5 μg mAb/million cells recommendation for TNF antibodies), as antibody concentration can significantly impact signal-to-noise ratios .

How can spectral overlap be managed when using TNN Antibody, FITC conjugated in multicolor flow cytometry?

Managing spectral overlap is critical when incorporating FITC-conjugated antibodies into multicolor panels. The emission spectrum of FITC (peak ~525nm) overlaps significantly with several other commonly used fluorophores:

• Compensation strategy:

  • Perform proper compensation using single-stained controls for each fluorophore

  • FITC significantly overlaps with PE (575nm) and requires careful compensation

  • Modern flow cytometers can automatically calculate the compensation matrix

• Panel design considerations:

  • Avoid pairing FITC with fluorophores having similar emission spectra (e.g., Alexa Fluor 488)

  • Reserve FITC for abundant antigens or strong signals

  • Place dimmer fluorophores on brighter markers and vice versa

• Alternative approaches:

  • When spectral overlap cannot be adequately compensated, consider using spectral unmixing algorithms

  • Sequential staining can be employed for complex panels where compensation is challenging

• Instrument-specific optimization:

  • FITC optimal excitation is at 495nm, typically excited with a 488nm laser

  • Filter selection should be optimized for the specific cytometer (typically 530/30 bandpass)

What methodological approaches can address nonspecific binding with TNN Antibody, FITC conjugated?

Nonspecific binding can compromise experimental data quality when using TNN Antibody, FITC conjugated. Several methodological approaches can mitigate this issue:

• Optimized blocking protocols:

  • Use 5-10% normal serum from the same species as the secondary antibody

  • Include 0.1-0.3% Triton X-100 for intracellular applications

  • BSA (1-3%) can reduce nonspecific hydrophobic interactions

• Sample preparation refinements:

  • Ensure complete fixation (typically 4% paraformaldehyde)

  • Optimize permeabilization conditions for intracellular targets

  • Remove dead cells, which often exhibit increased autofluorescence and nonspecific binding

• Titration optimization:

  • Determine the minimum antibody concentration that provides maximum specific signal

  • Typical starting range: 1-10 μg/mL, followed by serial dilutions to identify optimal concentration

• Pre-adsorption protocols:

  • Pre-adsorb the antibody with the target tissue lysate minus the specific antigen

  • This can significantly reduce cross-reactivity with unintended targets

• Buffer optimization:

  • Include 0.05-0.1% Tween-20 in wash buffers

  • Adjust salt concentration (150-300mM NaCl) to modify stringency

  • Consider pH adjustments based on the isoelectric point of the antibody

How can researchers validate TNN Antibody, FITC conjugated specificity for newly established experimental systems?

Validating antibody specificity is crucial when establishing new experimental systems. For TNN Antibody, FITC conjugated, a multi-faceted validation approach is recommended:

• Genetic validation:

  • Compare staining between wild-type cells and TNN knockout or knockdown systems

  • Overexpression systems can serve as positive controls

• Peptide competition assays:

  • Pre-incubate the antibody with excess recombinant Human Tenascin-N protein (441-556AA)

  • Specific staining should be significantly reduced or eliminated

• Orthogonal detection methods:

  • Confirm TNN expression using alternative methods (qPCR, Western blot)

  • Compare results from antibodies targeting different epitopes of TNN

• Cross-platform validation:

  • Compare flow cytometry results with immunofluorescence microscopy

  • Verify localization patterns match known TNN distribution

• Molecular weight confirmation:

  • For Western blot applications, confirm band size matches expected molecular weight

  • TNN has a theoretical molecular weight of approximately 160 kDa

• Positive and negative control tissues:

  • Use tissues with documented TNN expression patterns

  • Include tissues known to lack TNN expression as negative controls

What are the optimal fixation and permeabilization protocols for intracellular staining with TNN Antibody, FITC conjugated?

Intracellular staining with TNN Antibody, FITC conjugated requires careful consideration of fixation and permeabilization conditions to maintain both antigen recognition and fluorophore integrity:

• Fixation options:

  • Paraformaldehyde (2-4%): Preserves cellular architecture while maintaining most epitopes

  • Methanol/acetone: May enhance detection of some epitopes but can adversely affect FITC fluorescence

  • Hybrid protocols: Initial paraformaldehyde fixation followed by methanol permeabilization for certain applications

• Permeabilization strategies:

  • Saponin (0.1-0.5%): Gentle permeabilization suitable for most intracellular epitopes

  • Triton X-100 (0.1-0.3%): More stringent permeabilization for nuclear or tightly associated proteins

  • Digitonin (50 μg/mL): Selective permeabilization of plasma membrane while leaving organelle membranes intact

• Protocol optimization:

  • Temperature considerations: Room temperature fixation for 10-15 minutes is standard

  • Buffer composition: PBS with Ca²⁺/Mg²⁺ improves membrane preservation

  • Time optimization: Excessive fixation can mask epitopes through cross-linking

• Special considerations for FITC:

  • FITC fluorescence is pH-sensitive, optimize buffer pH (7.2-7.4)

  • Include antifade reagents to prevent photobleaching

  • Minimize exposure to light throughout the procedure

How should researchers approach quantification of TNN expression using FITC-conjugated antibodies?

Accurate quantification of TNN expression using FITC-conjugated antibodies requires standardized approaches:

• Flow cytometric quantification:

  • Use calibration beads with known FITC molecules of equivalent soluble fluorochrome (MESF)

  • Establish a standard curve to convert Mean Fluorescence Intensity (MFI) to molecules per cell

  • Calculate the antibody binding capacity (ABC) using appropriate calibration standards

• Microscopy-based quantification:

  • Implement consistent exposure settings across all samples

  • Use reference standards in each imaging session

  • Apply appropriate background subtraction methods

  • Consider photobleaching effects in time-course studies

• Standardization parameters:

  • Antibody lot consistency: Record lot numbers and validate new lots against previous standards

  • Instrument settings: Document PMT voltages, gain settings, and compensation matrices

  • Analysis templates: Establish and maintain consistent gating or region-of-interest strategies

• Dynamic range considerations:

  • FITC has a narrower dynamic range compared to some newer fluorophores

  • Avoid saturated signals, which prevent accurate quantification

  • Consider using a dilution series for highly expressed proteins

What approaches can resolve data inconsistencies when using TNN Antibody, FITC conjugated across different experimental platforms?

When facing inconsistencies across experimental platforms, systematic troubleshooting approaches can help identify and resolve issues:

• Sample preparation variables:

  • Standardize fixation timing, concentration, and temperature

  • Implement consistent blocking protocols

  • Control cell/tissue handling to minimize stress responses that may alter protein expression

• Technical standardization:

  • Use the same antibody lot across platforms when possible

  • Implement consistent antibody concentration and incubation conditions

  • Standardize washing steps (number, duration, buffer composition)

• Platform-specific optimization:

  • Flow cytometry: Optimize single-cell suspensions and gating strategies

  • Microscopy: Address issues of focal plane and 3D protein distribution

  • ELISA: Standardize coating conditions and blocking parameters

• Validation across platforms:

  • Use multiple antibodies against different TNN epitopes

  • Include spike-in controls of known concentration

  • Implement orthogonal measurement techniques

• Statistical approaches:

  • Implement normalization strategies appropriate to each platform

  • Use relative rather than absolute quantification when comparing across platforms

  • Apply appropriate statistical tests that account for platform-specific variance

How can researchers determine the optimal antibody concentration for specific applications?

Determining the optimal antibody concentration is critical for maximizing signal-to-noise ratio while minimizing reagent usage:

• Systematic titration approach:

  • Start with manufacturer's recommended concentration (if available)

  • Prepare a serial dilution series (typically 2-fold dilutions)

  • Test range from 0.1-10 μg/mL for most applications

  • For flow cytometry, follow similar principles to the ≤ 0.5 μg mAb/million cells recommendation for TNF antibodies

• Evaluation metrics:

  • Calculate signal-to-noise ratio at each concentration

  • Plot staining index (SI) = (MFI positive - MFI negative) / (2 × SD of negative population)

  • Determine the point of saturation (where increased concentration yields no improvement)

• Application-specific considerations:

  Application	Typical Concentration Range	Optimization Focus
  Flow cytometry	0.25-1 μg/mL	Separation index between positive and negative populations
  Immunofluorescence	1-5 μg/mL	Signal intensity vs. background
  ELISA	0.5-2 μg/mL	Standard curve linearity and dynamic range
  Western blot	0.2-1 μg/mL	Band specificity and background

• Economic considerations:

  • Determine the minimum concentration that provides reliable results

  • Balance antibody usage with experimental quality requirements

  • Consider the cost-benefit ratio of concentration vs. quality

How can TNN Antibody, FITC conjugated be utilized in multiplex imaging systems?

TNN Antibody, FITC conjugated can be effectively incorporated into multiplex imaging systems with appropriate planning:

• Spectral considerations:

  • FITC emission spectrum (peak ~525nm) must be separated from other fluorophores

  • Typical fluorophore combinations with minimal overlap include FITC, TRITC, Cy5, and DAPI

  • For higher multiplexing, consider spectral unmixing algorithms

• Sequential staining strategies:

  • Apply antibodies in order of increasing strength/brightness

  • Consider tyramide signal amplification (TSA) for weak signals

  • Implement antibody stripping between rounds for highly multiplexed approaches

• Multiplexing platforms:

  • Confocal microscopy: Optimized for 4-5 color separation

  • Spectral imaging: Can resolve closely overlapping fluorophores

  • Cyclic immunofluorescence: Allows for 20+ markers on the same section

• Cross-reactivity mitigation:

  • Use antibodies raised in different host species

  • Implement careful blocking between sequential staining steps

  • Consider fragment antibodies to reduce non-specific binding

• Image analysis considerations:

  • Implement channel-specific thresholding

  • Apply appropriate spectral unmixing algorithms

  • Consider machine learning approaches for complex pattern recognition

What are the critical parameters when developing flow cytometry panels incorporating TNN Antibody, FITC conjugated?

Developing effective flow cytometry panels incorporating TNN Antibody, FITC conjugated requires careful consideration of several parameters:

• Panel design principles:

  • Place FITC on markers with moderate to high expression

  • Avoid pairing FITC with PE due to significant spectral overlap

  • Consider protein co-expression patterns to maximize separation

• Technical optimization:

  • Voltage setting: Establish optimal PMT voltage for FITC channel

  • Compensation: Prepare single-stained controls for accurate compensation matrix

  • Fluorescence minus one (FMO) controls: Critical for setting accurate gates

• Staining protocol refinements:

  • Buffer optimization: Include protein blockers to reduce nonspecific binding

  • Incubation conditions: Standardize temperature and duration

  • Washing steps: Determine optimal number and composition

• Panel complexity considerations:

  Panel Complexity	Recommended Approach	Technical Considerations
  2-4 colors	Direct conjugates	Minimal compensation needed
  5-8 colors	Brightness matching	Critical compensation required
  >8 colors	Spectral cytometry	Unmixing algorithms instead of compensation

• Analysis strategy:

  • Implement consistent gating strategies

  • Consider dimensionality reduction techniques (tSNE, UMAP)

  • Analyze co-expression patterns systematically

How can researchers extend the fluorescence lifetime and stability of FITC-conjugated antibodies in long-term imaging experiments?

Long-term imaging experiments with FITC-conjugated antibodies present challenges due to photobleaching. Several strategies can extend fluorescence lifetime and stability:

• Antifade formulations:

  • p-Phenylenediamine (PPD): Effective but toxic and unstable

  • ProLong antifade reagents: Commercial solutions with superior performance

  • Vitamin C (ascorbic acid): Cost-effective alternative at 100mM concentration

• Imaging parameter optimization:

  • Reduce excitation intensity to minimum required

  • Implement interval scanning rather than continuous illumination

  • Use neutral density filters to attenuate excitation light

• Sample preparation refinements:

  • Rigorous elimination of reactive oxygen species

  • pH optimization (FITC is optimally fluorescent at pH 8-9)

  • Oxygen scavenging systems (e.g., glucose oxidase/catalase)

• Technical alternatives:

  • Consider photo-switchable fluorophores for super-resolution applications

  • Implement image acquisition strategies that minimize total light exposure

  • Use computational approaches to extract data from lower intensity images

• Storage considerations:

  • Maintain samples at 4°C when not imaging

  • Protect from light exposure between imaging sessions

  • Consider mounting media specifically formulated for long-term fluorescence preservation

What approaches can be used to validate colocalization of TNN with other proteins using FITC-conjugated antibodies?

• Quantitative colocalization metrics:

  • Pearson's correlation coefficient: Measures linear correlation between fluorophores

  • Manders' overlap coefficient: Proportion of overlapping pixels

  • Object-based colocalization: Analysis of discrete structures rather than pixels

• Technical controls for colocalization studies:

  • Single-labeled controls to assess bleed-through

  • Negative biological controls (proteins known not to colocalize)

  • Positive biological controls (proteins known to interact)

• Advanced imaging approaches:

  • Super-resolution microscopy (STED, STORM, SIM) to overcome diffraction limit

  • Förster resonance energy transfer (FRET) for direct interaction assessment

  • Live-cell imaging to observe dynamic colocalization

• Statistical validation:

  • Randomization tests to establish significance of observed colocalization

  • Analysis across multiple cells and experiments

  • Quantification of colocalization in specific subcellular compartments

• Complementary biochemical validation:

  • Co-immunoprecipitation to confirm physical interaction

  • Proximity ligation assay (PLA) to validate close spatial association

  • CRISPR-based tagging for endogenous protein localization

What emerging technologies are enhancing the applications of FITC-conjugated antibodies?

Recent technological advances are expanding the utility of FITC-conjugated antibodies:

• Advanced microscopy innovations:

  • Adaptive optics for deeper tissue imaging

  • Light sheet microscopy for rapid 3D acquisition

  • Expansion microscopy for physical sample enlargement

• Single-cell analysis integration:

  • Imaging mass cytometry combining spatial information with high-parameter analysis

  • Single-cell sequencing paired with index sorting

  • Digital spatial profiling for neighborhood context

• Computational advancements:

  • Deep learning for image restoration and noise reduction

  • Automated segmentation and quantification

  • Multi-dimensional data integration platforms

• New conjugation chemistries:

  • Site-specific conjugation for improved functionality

  • Environmentally responsive fluorophores

  • Multiplexed labeling strategies with orthogonal chemistries

• In vivo applications:

  • Intravital microscopy with stabilized FITC derivatives

  • Minimally invasive endoscopic technologies

  • Fluorescence-guided surgical applications