PIRT Antibody, FITC conjugated

What is PIRT antibody and what cellular structures does it recognize?

PIRT antibody recognizes phosphoinositide interacting regulator of TRP (PIRT), a membrane protein expressed specifically in the peripheral nervous system (PNS), predominantly in nociceptive (pain) neurons. The protein functions as a key component of the TRPV1 complex and positively regulates TRPV1 activity, which serves as a molecular sensor of pain . The antibody can detect the human PIRT protein in various experimental applications and is particularly useful for studying pain signaling pathways in neuronal tissues .

What is the significance of FITC conjugation for PIRT antibody applications?

FITC (fluorescein isothiocyanate) conjugation enables direct visualization of the PIRT protein in tissue sections or cultured cells through fluorescence microscopy. FITC is a small organic molecule that covalently binds to proteins via primary amines (i.e., lysines) . When conjugated to PIRT antibodies, it allows researchers to detect PIRT expression without requiring secondary antibodies, simplifying experimental procedures and potentially reducing background noise. FITC is typically excited by the 488 nm line of an argon laser, and emission is collected at 530 nm, making it compatible with standard fluorescence microscopy systems .

How should FITC-conjugated PIRT antibody be stored to maintain its activity?

FITC-conjugated antibodies, including PIRT antibody, should be stored protected from light as continuous exposure will cause gradual loss of fluorescence . The recommended storage condition is at -20°C or -80°C, avoiding repeated freeze-thaw cycles that can degrade antibody quality . The antibody is typically supplied in a protective buffer (containing 0.03% Proclin 300, 50% Glycerol, 0.01M PBS, pH 7.4) that helps maintain stability during storage . When handling the antibody, it should be kept in amber vials or wrapped in aluminum foil to protect from light exposure, and aliquoting is recommended to avoid repeated freeze-thaw cycles .

What are appropriate positive and negative controls for PIRT antibody experiments?

For positive controls, researchers should use tissues or cell lines known to express PIRT, particularly dorsal root ganglia (DRG) neurons, where PIRT is predominantly expressed . Studies have shown that PIRT is expressed in 83.9% of all DRG neurons, with particularly high expression in CGRP+ and IB4+ neuronal subtypes . For negative controls, researchers can use tissues from PIRT knockout mice (Pirt-/-), which show no detectable PIRT expression while maintaining normal proportions of neuronal subtypes in DRG . Additionally, using non-neuronal tissues that do not express PIRT or performing antibody preabsorption with recombinant PIRT protein can serve as technical negative controls.

How can I optimize double immunofluorescence protocols with PIRT-FITC antibody and other neuronal markers?

When performing double immunofluorescence with PIRT-FITC antibody and other neuronal markers, sequential staining protocols often yield better results than simultaneous staining. Begin with a thorough blocking step using PBS containing 10% fetal bovine serum for 20 minutes at room temperature to reduce non-specific binding . For double labeling with markers such as CGRP, IB4, or NF200 (which partially overlap with PIRT expression), apply the PIRT-FITC antibody first at a 1:500 dilution and incubate for 1 hour at room temperature in the dark . After washing with PBS, apply the second primary antibody (if directly conjugated) or use an unconjugated primary followed by a secondary antibody with a fluorophore spectrally distinct from FITC (e.g., Cy3, Cy5, or Alexa Fluors). Ensure cross-reactivity is minimized by using antibodies raised in different host species or by implementing additional blocking steps between applications.

What factors might affect PIRT antibody staining intensity and how can they be addressed?

Several factors can influence PIRT antibody staining intensity:

• Fixation method and duration: Overfixation can mask epitopes while underfixation may not adequately preserve tissue morphology. Optimize fixation protocols specifically for PIRT detection.

• Antigen retrieval: Although not specifically mentioned for PIRT antibody, many antibodies benefit from heat-induced epitope retrieval methods if initial staining is weak.

• Permeabilization efficiency: Since PIRT is a membrane protein, adequate permeabilization with detergents like Triton X-100 or saponin is crucial for antibody access.

• Antibody concentration: Titrate the antibody to find the optimal concentration that maximizes specific signal while minimizing background.

• Incubation conditions: Temperature and duration can affect binding kinetics; overnight incubation at 4°C may improve signal compared to shorter incubations at room temperature.

• Photobleaching: FITC is susceptible to photobleaching, so minimize exposure to light during preparation and imaging, and consider using anti-fade mounting media .

To address weak staining, increasing antibody concentration, extending incubation time, optimizing permeabilization, or employing signal amplification techniques like tyramide signal amplification might help improve detection sensitivity.

How can I quantitatively analyze PIRT expression patterns in neuronal subtypes?

For quantitative analysis of PIRT expression across neuronal subtypes:

• Collect z-stack images using confocal microscopy to capture the complete cellular distribution of PIRT.

• Implement co-labeling with established neuronal subtype markers: CGRP for peptidergic nociceptors, IB4 for non-peptidergic nociceptors, and NF200 for myelinated neurons .

• Use automated image analysis software (ImageJ/FIJI, CellProfiler, etc.) to:

  • Segment individual neurons based on morphological parameters

  • Measure fluorescence intensity of PIRT-FITC in each identified neuron

  • Classify neurons based on co-expression of subtype markers

  • Calculate the percentage of PIRT-positive cells within each neuronal subtype

• For quantitative comparison, normalize PIRT-FITC fluorescence intensity to account for variability between experiments, possibly using internal control markers or reference standards.

• Establish threshold values for positive versus negative staining based on control samples, including tissues from PIRT knockout animals .

Research has shown that PIRT is expressed in most CGRP+ and IB4+ neurons, with partial overlap in NF200+ myelinated neurons . Quantitative analysis can reveal differential expression patterns across these subtypes and potential changes in disease models or experimental interventions.

What are the technical considerations when using PIRT-FITC antibody in neuronal cultures versus tissue sections?

Neuronal Cultures:

• Fixation should be gentler for cultured neurons (e.g., 4% PFA for 10-15 minutes) to preserve fine neuronal processes.

• Background fluorescence is often lower in cultures, allowing for higher antibody dilutions (potentially 1:500-1:1000).

• Permeabilization should be carefully optimized as excessive detergent can damage delicate cultured neurons.

• Cultures provide better spatial resolution for subcellular localization studies of PIRT.

• Blocking solution with 10% FBS is typically sufficient to reduce non-specific binding .

Tissue Sections:

• Require more robust fixation and often benefit from antigen retrieval techniques.

• May have higher autofluorescence, particularly in the FITC channel, potentially requiring additional quenching steps.

• Thicker sections may need longer antibody incubation times or higher concentrations to ensure adequate penetration.

• May require more extensive washing steps to reduce background.

• When working with frozen tissue sections, the preservation of PIRT epitopes may be better than in paraffin sections .

In both applications, careful attention to controls is essential. For tissue sections, include adjacent sections stained with isotype control antibodies. For cultures, include wells where the primary antibody is omitted to assess secondary antibody specificity and background fluorescence.

How can PIRT antibody be used to investigate TRPV1-Pirt interactions in pain signaling pathways?

To investigate TRPV1-Pirt interactions in pain signaling pathways:

• Co-immunoprecipitation assays: Use PIRT antibody to pull down protein complexes from neuronal lysates and probe for TRPV1, or vice versa, to confirm physical interaction. This can be combined with treatments that modulate pain signaling to assess dynamic changes in the interaction.

• Proximity ligation assay (PLA): Employ PIRT-FITC antibody alongside TRPV1 antibody in a PLA protocol to visualize and quantify protein-protein interactions at subcellular resolution in intact neurons.

• Functional assays with calcium imaging: Apply PIRT antibody in live-cell calcium imaging experiments to determine if antibody binding modulates TRPV1 calcium influx in response to capsaicin or other TRPV1 agonists.

• Comparative analysis in WT vs. Pirt-/- neurons: Use the antibody to quantify TRPV1 membrane localization and function in wild-type versus Pirt knockout neurons to determine how PIRT regulates TRPV1 trafficking and activity .

• Phosphoinositide binding studies: Since PIRT is a phosphoinositide-binding protein, employ PIRT antibody in assays that examine how phosphoinositides influence PIRT-TRPV1 interactions and subsequent channel modulation .

Research indicates that PIRT is a key component of the TRPV1 complex and positively regulates TRPV1 activity . Understanding this interaction is crucial for developing targeted pain therapeutics.

What are common problems with FITC-conjugated antibodies and their solutions?

Problem: Rapid photobleaching during imaging

• Solution: Minimize exposure time during imaging, use anti-fade mounting media, capture FITC channel images first in multi-channel experiments, and consider using newer generation fluorophores (Alexa Fluor 488) for follow-up experiments if photobleaching is severe .

Problem: High background fluorescence

• Solution: Increase blocking time using PBS with 10% FBS, optimize antibody dilution (typically start at 1:500), extend wash steps, and consider using specific blocking reagents for endogenous biotin or Fc receptors if present in your sample .

Problem: Weak or absent signal

• Solution: Verify PIRT expression in your sample type, optimize fixation and permeabilization protocols, try lower antibody dilutions, extend incubation time, and ensure proper storage of the antibody to maintain fluorescence activity .

Problem: Non-specific binding

• Solution: Increase blocking time and concentration, perform more thorough washing steps, and validate specificity with appropriate controls including PIRT knockout tissues or competitive blocking with the immunizing peptide .

Problem: Autofluorescence masking specific signal

• Solution: Include an autofluorescence quenching step in your protocol, such as treatment with sodium borohydride or copper sulfate, or use spectral unmixing during image acquisition if your microscope system supports it.

How should I design experiments to validate PIRT antibody specificity?

A comprehensive validation strategy for PIRT antibody should include:

• Genetic validation: Compare staining between wild-type and Pirt knockout (Pirt−/−) tissues or cells, where the antibody should show positive staining in wild-type samples and no specific staining in knockout samples .

• Peptide competition assay: Pre-incubate the antibody with excess immunizing peptide (recombinant Human Phosphoinositide-interacting protein, amino acids 1-55) before applying to samples; specific staining should be blocked .

• Western blot analysis: Confirm the antibody detects a band of the expected molecular weight for PIRT in lysates from tissues known to express the protein.

• Correlation with mRNA expression: Perform in situ hybridization with Pirt riboprobe on serial sections to correlate protein detection with mRNA expression patterns .

• Cross-reactivity testing: Test the antibody on tissues from multiple species if cross-reactivity is claimed, or on tissues known not to express PIRT as negative controls.

• Reproducibility assessment: Verify consistent staining patterns across multiple experimental replicates and different sample preparation methods.

Research has shown that Pirt is expressed in 83.9% of all DRG neurons, with particularly high expression in CGRP+ and IB4+ neurons, providing a reference pattern for validation studies .

What protocol modifications are needed for double immunofluorescence with PIRT-FITC and other conjugated antibodies?

When performing double immunofluorescence with PIRT-FITC and other directly conjugated antibodies, several protocol modifications are necessary:

• Fluorophore selection: Choose secondary fluorophores with minimal spectral overlap with FITC (emission peak at 530 nm). Good companions include Cy3, Cy5, Alexa Fluor 594, or Alexa Fluor 647.

• Sequential staining approach:

  • First complete the staining with PIRT-FITC antibody (dilution 1:500 in PBS/10% FBS, incubate for 1 hour at room temperature in the dark)

  • Wash thoroughly with PBS (3-5 times, 5 minutes each)

  • Apply additional blocking step with serum matching the host species of the second primary antibody

  • Proceed with the second primary antibody staining

  • Wash and mount with anti-fade medium

• Cross-reactivity prevention: If both primary antibodies are from the same host species (e.g., both rabbit), implement additional blocking steps using unconjugated Fab fragments against the first primary antibody before applying the second antibody.

• Imaging controls: Include single-stained controls for each antibody to set proper imaging parameters and confirm lack of bleed-through between channels.

• Signal balancing: Adjust antibody concentrations individually to achieve comparable signal intensities, as FITC may be less bright than some newer fluorophores.

The protocol should be validated by comparing the staining pattern with previously published data showing PIRT expression in CGRP+, IB4+, and some NF200+ neurons in the DRG .

How can I optimize PIRT antibody staining for challenging neuronal tissue samples?

For challenging neuronal tissue samples:

• Fixation optimization:

  • Try shorter fixation times (4-8 hours) with 4% PFA rather than overnight fixation

  • Consider alternative fixatives such as methanol or acetone for improved epitope preservation

  • For highly myelinated tissues, include a brief post-fixation permeabilization step with methanol

• Enhanced permeabilization:

  • For thick sections or heavily myelinated tissue, increase Triton X-100 concentration (up to 0.3-0.5%)

  • Consider using saponin (0.1-0.2%) which can be gentler on membrane proteins like PIRT

  • Implement a freeze-thaw cycle to improve antibody penetration in thick sections

• Antigen retrieval methods:

  • Heat-induced epitope retrieval using citrate buffer (pH 6.0)

  • Enzymatic retrieval with proteases like proteinase K (use with caution as it may damage tissue morphology)

  • Try a combination of heat and enzymatic methods for particularly challenging samples

• Signal amplification:

  • Employ tyramide signal amplification (TSA) to enhance FITC signal

  • Use biotin-streptavidin amplification systems prior to FITC detection

  • Consider sequential application of primary and secondary antibodies with amplification steps

• Background reduction:

  • Extended blocking (2+ hours or overnight) with PBS/10% FBS with addition of 0.1-0.3% Triton X-100

  • Include 0.1-0.3% BSA and 0.1% fish gelatin in blocking buffer to reduce non-specific binding

  • Pre-absorb the antibody with tissue homogenates from non-expressing tissues

These optimizations should be systematically tested and documented to establish a reliable protocol for detecting PIRT in challenging tissue samples.

What standards and quantitative methods should be used to report PIRT expression levels?

For standardized reporting of PIRT expression levels:

• Quantitative fluorescence measurements:

  • Mean fluorescence intensity (MFI) of positively stained cells compared to background

  • Integrated density values that account for both intensity and area of staining

  • Signal-to-noise ratio calculations using unstained areas as reference

• Standardization controls:

  • Include calibration standards with known fluorophore concentrations in each experiment

  • Use reference tissues with established PIRT expression levels for inter-experimental normalization

  • Include wild-type and Pirt-/- samples as positive and negative controls

• Cell counting methods:

  • Report percentage of PIRT-positive cells within specific neuronal populations (e.g., 83.9% of all DRG neurons express PIRT)

  • Use automated counting algorithms with consistent thresholding parameters

  • Implement double-blind counting to reduce observer bias

• Co-localization analysis:

  • Pearson's or Mander's correlation coefficients for co-localization with other markers

  • Nearest neighbor analysis for spatial relationships between PIRT+ and other cell types

  • Line profile analysis across cell compartments for subcellular distribution

• Statistical reporting standards:

  • Minimum sample sizes: analyze at least 100-200 neurons per condition

  • Include biological replicates (different animals/tissue samples) and technical replicates

  • Report data as mean ± SEM with appropriate statistical tests for comparisons

• Data presentation:

  • Use consistent pseudocoloring in images (typically green for FITC)

  • Include scale bars on all images

  • Present quantitative data in graphs alongside representative images

These standardized approaches will enable more reliable comparisons of PIRT expression across different studies and experimental conditions.

How can PIRT-FITC antibody be used to study pain mechanisms and neuropathic conditions?

PIRT-FITC antibody offers several advantages for studying pain mechanisms and neuropathic conditions:

• Monitoring expression changes in pain models:

  • Quantify changes in PIRT expression levels in various neuropathic pain models (nerve injury, inflammation, diabetic neuropathy)

  • Track temporal expression patterns during pain development and resolution

  • Compare PIRT distribution in affected versus unaffected neurons within the same ganglion

• Investigating TRPV1-PIRT interactions in pain states:

  • Evaluate co-localization changes between PIRT and TRPV1 under pathological conditions

  • Assess whether PIRT translocation occurs during sensitization processes

  • Determine if PIRT-TRPV1 interaction is altered by analgesic treatments

• Therapeutic target validation:

  • Use PIRT-FITC antibody to screen compounds that might disrupt or enhance PIRT-TRPV1 interactions

  • Validate the effects of PIRT-targeting therapies on protein expression and localization

  • Monitor changes in PIRT expression following treatment with established analgesics

• Functional correlation studies:

  • Combine PIRT immunofluorescence with calcium imaging or electrophysiology to correlate expression levels with functional responses

  • Identify neuronal subpopulations where PIRT expression correlates with hyperexcitability in pain states

  • Link PIRT expression patterns to behavioral pain outcomes in animal models

Since PIRT is expressed in 83.9% of all DRG neurons and is a key regulator of TRPV1, a molecular sensor of pain, these applications could significantly advance our understanding of pain mechanisms and lead to novel therapeutic approaches .

What emerging techniques can be combined with PIRT-FITC immunofluorescence for advanced research?

Emerging techniques that can be integrated with PIRT-FITC immunofluorescence include:

• Expansion microscopy (ExM):

  • Physically expanding specimens to achieve super-resolution imaging with standard confocal microscopes

  • Allows visualization of nanoscale co-localization between PIRT and interaction partners

  • Particularly valuable for examining PIRT distribution in fine neuronal processes

• Tissue clearing techniques:

  • CLARITY, CUBIC, or iDISCO methods for whole-tissue immunolabeling and imaging

  • Enables 3D visualization of PIRT expression throughout entire ganglia or nerve segments

  • Preserves spatial relationships between PIRT+ neurons and surrounding structures

• Single-cell transcriptomics correlation:

  • Combining PIRT immunofluorescence with RNAscope or similar in situ hybridization techniques

  • Correlating protein expression with transcriptomic profiles at single-cell resolution

  • Identifying transcriptional signatures associated with varying PIRT expression levels

• Optogenetic integration:

  • Pairing PIRT-FITC labeling with optogenetic markers in specific neuronal populations

  • Correlating PIRT expression with functional responses to optogenetic stimulation

  • Investigating how PIRT levels affect neuronal excitability in precisely defined circuits

• Live-cell antibody-based imaging:

  • Using membrane-permeable fluorescently labeled antibody fragments to track PIRT dynamics in living neurons

  • Monitoring real-time changes in PIRT localization during nociceptive signaling

  • Observing PIRT-phosphoinositide interactions in response to cellular stimulation

These integrative approaches can provide unprecedented insights into PIRT's role in neuronal signaling and pain sensation, potentially revealing new therapeutic targets for pain management.

How does PIRT expression compare across different species and what are the implications for translational research?

While the search results don't provide comprehensive cross-species data, we can discuss important considerations for translational research:

Understanding species differences in PIRT expression is critical for developing targeted pain therapies that successfully translate from preclinical models to human applications.

What are the most effective imaging parameters for FITC when analyzing PIRT localization at subcellular resolution?

For optimal imaging of PIRT-FITC at subcellular resolution:

• Excitation/Emission parameters:

  • Excitation maximum: 488-495 nm (typically using argon laser or similar LED/filter sets)

  • Emission collection: 510-530 nm with narrow bandpass filters to minimize bleed-through

  • Use spectral detection systems if available to optimize signal separation

• Confocal microscopy settings:

  • Pinhole: 1 Airy unit for optimal resolution/signal balance; reduce to 0.7-0.8 for higher resolution

  • Line averaging: 4-8× to improve signal-to-noise ratio

  • Pixel dwell time: Longer dwell times improve signal but increase photobleaching; find optimal balance

  • Z-step size: 0.3-0.5 μm for Nyquist sampling at diffraction-limited resolution

  • Sequential scanning when using multiple fluorophores to prevent crosstalk

• Super-resolution approaches:

  • Stimulated Emission Depletion (STED) microscopy: Can achieve 30-80 nm resolution with FITC

  • Structured Illumination Microscopy (SIM): Provides ~100 nm resolution, good for FITC with less photobleaching than STED

  • Single Molecule Localization Microscopy (PALM/STORM): Requires specialized photoconvertible fluorophores, consider antibody re-labeling strategies

• Sample preparation considerations:

  • Mounting medium: Use anti-fade agents specifically optimized for FITC to reduce photobleaching

  • Cover glass thickness: Use #1.5 (0.17 mm) for optimal optical properties

  • Refractive index matching: Ensure mounting medium matches objective immersion medium

• Quantitative imaging controls:

  • Include fluorescent beads as intensity standards

  • Apply flat-field correction to account for illumination non-uniformities

  • Use blind deconvolution to improve resolution while maintaining quantitative integrity

FITC can readily achieve diffraction-limited resolution (~250 nm laterally, ~700 nm axially) with standard confocal microscopy and can be pushed to super-resolution with appropriate techniques .

How can PIRT-FITC antibody contribute to drug discovery for pain management?

PIRT-FITC antibody can significantly contribute to drug discovery for pain management through several approaches:

• High-content screening applications:

  • Develop cell-based assays measuring PIRT-TRPV1 interaction disruption by candidate compounds

  • Screen for drugs that modulate PIRT membrane localization or expression levels

  • Identify compounds that alter PIRT's interaction with phosphoinositides, which regulate its activity

• Target validation studies:

  • Confirm that drug candidates engaging PIRT pathway components produce expected changes in protein localization or expression

  • Visualize on-target effects at the cellular level in relevant neuronal populations

  • Track compensatory changes in PIRT expression following chronic drug administration

• Mechanistic investigations:

  • Elucidate precise molecular mechanisms by which lead compounds affect PIRT-dependent signaling

  • Determine whether effective analgesics alter PIRT expression patterns in pain models

  • Identify which neuronal subtypes show PIRT modulation in response to effective treatments

• Translational biomarker development:

  • Establish whether changes in PIRT expression correlate with pain states in preclinical models

  • Determine if PIRT expression pattern changes could serve as pharmacodynamic biomarkers

  • Develop protocols for monitoring PIRT in accessible human tissues (e.g., skin biopsies) as potential clinical biomarkers

• Combination therapy rational design:

  • Identify synergistic targets by examining effects of combination treatments on PIRT and its signaling partners

  • Visualize pathway-specific effects to minimize off-target actions

  • Optimize dosing regimens based on temporal changes in PIRT expression and localization

Given that PIRT is a key component of the TRPV1 complex and positively regulates TRPV1 activity, a molecular sensor of pain, targeting this pathway has significant therapeutic potential for developing novel analgesics with improved efficacy and reduced side effects .

What is the detailed protocol for PIRT-FITC antibody staining in frozen tissue sections?

Protocol for PIRT-FITC Antibody Staining in Frozen Tissue Sections

Materials Required:

• PIRT-FITC conjugated antibody (e.g., QA50220)

• PBS (0.01M, pH 7.4)

• Blocking solution (PBS containing 10% fetal bovine serum)

• 0.1-0.3% Triton X-100 in PBS

• Anti-fade mounting medium

• Glass slides and coverslips

Procedure:

• Tissue Preparation:

  • Cut frozen tissue sections at 10-15 μm thickness

  • Air-dry sections for 30 minutes at room temperature

  • Fix sections in cold acetone or 4% paraformaldehyde for 10 minutes

  • Wash 3 times with PBS, 5 minutes each

• Permeabilization:

  • Incubate sections in 0.1-0.3% Triton X-100 in PBS for 10 minutes at room temperature

  • Wash 3 times with PBS, 5 minutes each

• Blocking:

  • Add blocking solution (PBS containing 10% fetal bovine serum) and incubate for 20 minutes at room temperature

  • Remove blocking solution without washing

• Primary Antibody Incubation:

  • Dilute PIRT-FITC antibody 1:500 in PBS/10% FBS

  • Apply 100-200 μl per section

  • Incubate in a humidified chamber for 1 hour at room temperature in the dark

  • For enhanced sensitivity, incubate overnight at 4°C in the dark

• Washing:

  • Wash sections 3 times with PBS, 5 minutes each, protecting from light

• Counterstaining (optional):

  • Incubate with DAPI (1 μg/ml in PBS) for 5 minutes to visualize nuclei

  • Wash once with PBS for 5 minutes

• Mounting:

  • Mount sections with anti-fade mounting medium

  • Seal edges with nail polish

  • Store slides at 4°C in the dark

• Imaging:

  • Visualize using a fluorescence microscope with appropriate FITC filter set (excitation ~490 nm, emission ~525 nm)

  • Capture images promptly to minimize photobleaching

Controls:

• Include a section stained with isotype control antibody conjugated to FITC

• Include a section with primary antibody omitted to assess autofluorescence

This protocol is based on standard immunofluorescence procedures and recommendations for FITC-conjugated antibodies .

What buffer conditions optimize PIRT-FITC antibody performance and stability?

The optimal buffer conditions for PIRT-FITC antibody performance and stability include:

Storage Buffer:

• Preservative: 0.03% Proclin 300

• Constituents: 50% Glycerol, 0.01M PBS, pH 7.4

• Storage temperature: -20°C or -80°C

Working Buffer Recommendations:

• Dilution Buffer:

  • PBS containing 10% fetal bovine serum (optimal for reducing background)

  • pH 7.4 (critical for FITC fluorescence stability)

  • Optional addition of 0.1% Triton X-100 for improved penetration in tissue sections

• Wash Buffer:

  • PBS with 0.05-0.1% Tween-20

  • Maintain pH between 7.2-7.4 for optimal FITC fluorescence

• pH Considerations:

  • FITC fluorescence is pH-sensitive, with optimal emission at slightly alkaline pH (7.4-8.0)

  • Avoid acidic conditions (below pH 7.0) which can significantly reduce fluorescence intensity

• Stabilizing Additives:

  • Addition of 1-5% BSA can improve antibody stability during storage and incubation

  • 0.01-0.05% sodium azide can prevent microbial growth in stored working dilutions (avoid if using enzyme-based detection systems)

• Light Protection:

  • Amber tubes or aluminum foil wrapping for all solutions containing the FITC conjugate

  • Minimize exposure to light during all steps

• Antioxidant Addition:

  • 0.1-1 mM sodium ascorbate can be added to working solutions to reduce photobleaching

  • 5-10 mM n-propyl gallate in mounting media improves FITC signal durability

• Temperature Considerations:

  • Perform all dilutions with pre-cooled buffers (4°C)

  • Allow solutions to equilibrate to room temperature before applying to specimens

These buffer conditions help maintain the structural integrity of the antibody while preserving FITC fluorescence properties for optimal staining results .

What is the comparative data on PIRT expression across neuronal subtypes?

Based on the search results, the following comparative data summarizes PIRT expression across neuronal subtypes:

Table 1: PIRT Expression in Dorsal Root Ganglion (DRG) Neuronal Subtypes

Key Observations:

• PIRT is expressed in the majority (83.9%) of DRG neurons, suggesting a fundamental role in sensory neuron function .

• PIRT expression is particularly high in nociceptive neurons (both CGRP+ and IB4+ populations) .

• There is partial overlap between PIRT expression and NF200+ myelinated neurons, indicating potential functions beyond nociception .

• TRPV1+ neuron percentages are comparable between wild-type and Pirt knockout mice, suggesting that PIRT does not regulate TRPV1 expression but rather modulates its function .

• The immunoreactivity and projection patterns of TRPV1+, CGRP+, and IB4+ primary afferents to the spinal cord are similar between wild-type and Pirt knockout mice, indicating PIRT is not involved in the development of nociceptive neurons .

This expression pattern underscores PIRT's importance in pain signaling pathways and suggests it may have distinct functional roles across different neuronal subtypes.

What methods should be used to quantify the number of FITC molecules conjugated to each PIRT antibody?

To accurately quantify the number of FITC molecules conjugated to each PIRT antibody (degree of labeling or DOL), researchers should employ the following methods:

• Spectrophotometric Method:

  • Measure absorbance at 280 nm (A₂₈₀) for protein concentration and 495 nm (A₄₉₅) for FITC

  • Calculate DOL using the formula:
    DOL = (A₄₉₅ × ε_protein) / [(A₂₈₀ - A₄₉₅ × CF) × ε_FITC]
    Where:

    • ε_protein = molar extinction coefficient of antibody (typically ~210,000 M⁻¹cm⁻¹ for IgG)

    • ε_FITC = molar extinction coefficient of FITC (typically ~68,000-75,000 M⁻¹cm⁻¹)

    • CF = correction factor for FITC contribution at 280 nm (typically 0.3-0.35)

  • Optimal conjugation typically yields 3-6 FITC molecules per antibody

• Fluorescence Standard Curve Method:

  • Prepare a standard curve using free FITC of known concentrations

  • Measure fluorescence intensity of the conjugated antibody

  • Interpolate the amount of FITC from the standard curve

  • Divide by the molar concentration of antibody to determine average DOL

• MALDI-TOF Mass Spectrometry:

  • Compare the molecular weight of unconjugated antibody with FITC-conjugated antibody

  • Calculate the difference in mass and divide by the molecular weight of the FITC-reactive group (~389 Da)

  • This provides a precise measurement of average DOL

• Size-Exclusion HPLC:

  • Run both unconjugated and conjugated antibody on SEC-HPLC

  • Monitor absorbance at both 280 nm and 495 nm

  • Calculate the ratio of FITC to protein based on peak areas and extinction coefficients

• Comparative Performance Testing:

  • Compare brightness of antibodies with different conjugation ratios in actual staining experiments

  • Optimal conjugation should maximize brightness while avoiding internal quenching and solubility problems that occur with excessive conjugation

Research indicates that usually between 3 and 6 FITC molecules are conjugated to each antibody; higher conjugations can result in solubility problems as well as internal quenching and reduced brightness . Therefore, when conjugating antibodies, multiple parallel reactions with different amounts of FITC should be performed, and the resulting conjugates compared for brightness and background binding to determine the optimal ratio .

What quality control parameters should be assessed before using PIRT-FITC antibody in critical experiments?

Before using PIRT-FITC antibody in critical experiments, researchers should assess the following quality control parameters:

• Conjugation Quality:

  • Degree of labeling (DOL): Verify that 3-6 FITC molecules are conjugated per antibody for optimal performance

  • Aggregation assessment: Confirm absence of precipitates or aggregates via visual inspection and/or dynamic light scattering

  • Free FITC content: Should be <5% of total fluorescence to minimize background

• Antibody Functionality:

  • Specific binding: Confirm reactivity to recombinant human phosphoinositide-interacting protein (amino acids 1-55)

  • Comparison to unconjugated version: Verify that FITC conjugation hasn't significantly impaired antigen recognition

  • Cross-reactivity testing: Check for non-specific binding to unrelated proteins

• Fluorescence Properties:

  • Brightness: Measure quantum yield relative to free FITC standard

  • Photobleaching rate: Assess stability under continuous illumination

  • pH sensitivity: Confirm consistent fluorescence across working pH range

• Application-Specific Validation:

  • Signal-to-noise ratio: Determine in relevant tissue types

  • Staining pattern: Compare with published PIRT localization data

  • Specificity controls: Test on Pirt knockout tissues if available

• Reagent Stability:

  • Freeze-thaw stability: Test performance after multiple freeze-thaw cycles

  • Storage duration: Verify activity retention over expected storage period

  • Light exposure sensitivity: Quantify fluorescence loss after defined light exposure

• Documentation Requirements:

  • Lot-to-lot consistency: Compare with previous lots if available

  • Expiration dating: Establish based on stability testing

  • Certificate of analysis: Review from supplier for key specifications

• Control Experiments:

  • Positive control: Test on tissues known to express PIRT (e.g., DRG neurons)

  • Negative control: Apply isotype control antibody with FITC at same concentration

  • Blocking experiment: Pre-incubate with immunizing peptide to confirm specificity