OR5H6 Antibody, FITC conjugated

What is OR5H6 and why is it significant in neuroscience research?

OR5H6 (Olfactory Receptor, Family 5, Subfamily H, Member 6) is a human olfactory receptor protein with UniProt accession number Q8NGV6. This G protein-coupled receptor plays a crucial role in olfactory signal transduction pathways. Researchers target OR5H6 to investigate olfactory perception mechanisms, neuronal signaling pathways, and potential associations with neurodegenerative disorders. The OR5H6 antibody allows visualization and quantification of this receptor in various experimental contexts, enabling the mapping of expression patterns across different neural tissues and developmental stages. The antibody specifically recognizes amino acids 277-292 of the human OR5H6 protein, a region selected for its antigenicity and specificity.

What are the optimal storage conditions for maximizing OR5H6 Antibody, FITC conjugated stability?

OR5H6 Antibody, FITC conjugated should be stored at -20°C or -80°C immediately upon receipt to maintain optimal reactivity. Repeated freeze-thaw cycles significantly reduce antibody efficacy and should be strictly avoided. The antibody is supplied in a buffer containing 50% glycerol and 0.03% Proclin 300 in 0.01M PBS (pH 7.4) to stabilize the protein and prevent microbial growth. For short-term storage (1-2 weeks), aliquoting into single-use volumes and storing at 4°C is acceptable, but prolonged storage at this temperature will accelerate the degradation of the FITC fluorophore, resulting in decreased signal intensity. Laboratory records should document storage conditions and freeze-thaw cycles to correlate with experimental outcomes.

What applications is the OR5H6 Antibody, FITC conjugated validated for?

The OR5H6 Antibody, FITC conjugated has been validated primarily for immunofluorescence (IF) applications in human tissue samples and cell lines expressing the target protein. While the antibody documentation recommends inquiring about specific applications, its FITC conjugation makes it particularly suited for direct immunofluorescence microscopy and flow cytometry analyses. The antibody's Protein G purification (>95% purity) ensures minimal background and non-specific binding. For Western blot applications, unconjugated variants of the OR5H6 antibody targeting the same epitope (amino acids 277-292) may provide better results, as some studies indicate that direct FITC conjugation can occasionally interfere with protein binding in denatured sample conditions. Experimental validation in the specific experimental system is always recommended before proceeding with large-scale studies.

What is the recommended protocol for immunofluorescence using OR5H6 Antibody, FITC conjugated?

For optimal immunofluorescence detection using OR5H6 Antibody, FITC conjugated, follow this validated protocol:

• Fix cells or tissue sections with 4% paraformaldehyde for 15 minutes at room temperature.

• Permeabilize with 0.2% Triton X-100 in PBS for 10 minutes (for intracellular epitopes).

• Block with 5% normal serum (from species not related to primary antibody) in PBS for 1 hour.

• Apply diluted OR5H6 Antibody, FITC conjugated (typically 1:20-1:100 dilution in blocking buffer) and incubate for 1-2 hours at room temperature or overnight at 4°C in a humidified chamber protected from light.

• Wash thoroughly with PBS (3 × 5 minutes).

• Counterstain nuclei with DAPI if desired.

• Mount with anti-fade mounting medium.

• Visualize using fluorescence microscopy with appropriate filters for FITC detection (excitation: 492nm, emission: 520nm).

This direct immunofluorescence approach eliminates the need for secondary antibody incubation, reducing background and potential cross-reactivity issues. Optimize antibody dilution for your specific sample type, as fixation methods can affect epitope accessibility.

How should I design proper controls for experiments using OR5H6 Antibody, FITC conjugated?

A comprehensive control strategy for OR5H6 Antibody, FITC conjugated experiments should include:

• Positive Control: Cells or tissues known to express OR5H6 protein (typically certain neural tissues).

• Negative Control: Samples lacking OR5H6 expression or knockdown/knockout models.

• Isotype Control: FITC-conjugated rabbit IgG at the same concentration as the OR5H6 antibody to assess non-specific binding.

• Autofluorescence Control: Unstained sample to establish baseline tissue autofluorescence.

• Absorption Control: Pre-incubating the antibody with excess recombinant OR5H6 protein (amino acids 277-292) before applying to samples should abolish specific staining.

• Secondary-only Control: Not applicable for direct FITC conjugates, but useful if parallel experiments with unconjugated antibodies are performed.

For quantitative analyses, include fluorescence calibration standards to normalize signal intensity across experimental runs. Document microscope settings (exposure time, gain, etc.) for reproducibility, and process all images using identical parameters to ensure valid comparisons.

What are the recommended approaches for co-localization studies involving OR5H6?

For co-localization studies involving OR5H6 Antibody, FITC conjugated and other cellular markers:

• Select companion antibodies raised in species other than rabbit to avoid cross-reactivity.

• Choose fluorophores with minimal spectral overlap with FITC (excitation 492nm/emission 520nm) - Cy3, Texas Red, or far-red dyes are excellent choices.

• Perform sequential staining protocol:

  • Complete the OR5H6 Antibody, FITC protocol first

  • Apply additional blocking step with 5% rabbit serum

  • Proceed with unconjugated primary antibody against second target

  • Use appropriate species-specific secondary antibody with compatible fluorophore

For quantitative co-localization analysis, apply rigorous statistical methods such as Pearson's correlation coefficient or Manders' overlap coefficient. Conduct photobleaching controls to ensure FITC signal stability during extended imaging sessions. Z-stack imaging with deconvolution is recommended for three-dimensional co-localization assessment to minimize false positive results from superimposed signals in different focal planes.

How can I optimize signal-to-noise ratio when using OR5H6 Antibody, FITC conjugated?

To maximize signal-to-noise ratio with OR5H6 Antibody, FITC conjugated:

• Sample preparation optimization:

  • Freshly prepared fixatives yield better results than stored solutions

  • Perform antigen retrieval if necessary (citrate buffer pH 6.0, 95°C for 20 minutes)

  • Extend blocking time to 2 hours with 5-10% serum plus 1% BSA

  • Include 0.1% Tween-20 in washing buffers to reduce non-specific binding

• Antibody optimization:

  • Titrate antibody concentration (typically start with 1:50 and adjust)

  • Extend incubation time at 4°C to increase specific binding

  • Filter antibody solution through 0.22μm filter before use to remove aggregates

• Imaging optimization:

  • Use narrow bandpass FITC filters to reduce autofluorescence

  • Apply appropriate image acquisition settings (avoid overexposure)

  • Consider confocal microscopy to improve signal-to-noise ratio

  • Apply digital deconvolution algorithms if appropriate

• Autofluorescence mitigation:

  • Treat sections with 0.1% Sudan Black B in 70% ethanol for 20 minutes before blocking

  • Consider spectral unmixing during image acquisition and processing

Document all optimization steps systematically to establish a reproducible protocol for your specific experimental system.

What are common sources of false positives/negatives when working with OR5H6 Antibody, FITC conjugated?

Each potential error source requires systematic troubleshooting. When encountering unexpected results, methodically alter a single variable at a time to identify the problematic step in your protocol.

How can I quantify OR5H6 expression levels using FITC-conjugated antibody?

For rigorous quantification of OR5H6 expression using FITC-conjugated antibody:

• Flow Cytometry Quantification:

  • Use calibration beads with known FITC molecules per bead to establish standard curve

  • Calculate Molecules of Equivalent Soluble Fluorochrome (MESF) values

  • Apply compensation if using multiple fluorophores

  • Report median fluorescence intensity (MFI) rather than mean values

• Microscopy-based Quantification:

  • Include fluorescence intensity calibration standards in each experiment

  • Capture all images using identical acquisition parameters

  • Apply flat-field correction to compensate for uneven illumination

  • Use specialized software (ImageJ/FIJI with appropriate plugins) for quantitative analysis

  • Define regions of interest (ROI) systematically using objective criteria

  • Report integrated density values normalized to area

• Validation Approaches:

  • Compare results with orthogonal methods (RT-qPCR, Western blot)

  • Establish dose-response curves with known modifiers of OR5H6 expression

  • Include biological replicates to account for natural variation

• Statistical Analysis:

  • Apply appropriate statistical tests based on data distribution

  • Report confidence intervals alongside means

  • Consider using non-parametric tests if normality assumptions are violated

These quantification approaches provide robust measurements that can be correlated across experimental conditions and between laboratories.

How can OR5H6 Antibody, FITC conjugated be adapted for super-resolution microscopy?

Adapting OR5H6 Antibody, FITC conjugated for super-resolution microscopy requires careful consideration of several technical factors:

• FITC Compatibility Assessment:

  • While FITC is not optimal for all super-resolution techniques, it can be used with Structured Illumination Microscopy (SIM) and certain Stimulated Emission Depletion (STED) setups

  • For Single Molecule Localization Microscopy (SMLM) techniques like STORM or PALM, consider using antibody labeled with more photostable fluorophores

• Sample Preparation Optimization:

  • Use thinner tissue sections (≤5μm) or flat cell monolayers

  • Increase fixation stringency to minimize sample movement

  • Mount samples on high-precision coverslips (#1.5H, 170±5μm thickness)

  • Use specialized mounting media with matched refractive index

• Imaging Protocol Adjustments:

  • Increase antibody concentration by 1.5-2× to ensure sufficient labeling density

  • Optimize buffer conditions to enhance FITC photostability

  • For STED microscopy, pair with appropriate depletion laser (592nm)

  • For SIM, ensure high signal-to-noise ratio through optimized blocking

• Validation Strategy:

  • Compare with conventional confocal microscopy results

  • Use fiducial markers for drift correction

  • Implement appropriate image reconstruction algorithms

  • Apply rigorous statistical analysis to super-resolution data

While direct FITC conjugates may have limitations for some super-resolution applications, newer generations of more photostable green fluorophores can be considered as alternatives for advanced imaging studies of OR5H6.

What are the considerations for using OR5H6 Antibody, FITC conjugated in neuronal tissue studies?

Working with OR5H6 Antibody, FITC conjugated in neuronal tissues presents unique challenges that require specialized approaches:

• Tissue-Specific Optimization:

  • Extend fixation time for brain tissue (24-48 hours in 4% PFA)

  • Perform antigen retrieval using citrate buffer (pH 6.0) or EDTA buffer (pH 8.0)

  • Use thicker sections (30-50μm) for structural studies, thinner sections (5-10μm) for high-resolution imaging

  • Consider vibratome sectioning for preserving tissue architecture

• Background Reduction Strategies:

  • Pre-treat sections with 1% H₂O₂ to quench endogenous peroxidase activity

  • Apply Sudan Black B (0.1% in 70% ethanol) to reduce lipofuscin autofluorescence

  • Include 0.1-0.3% Triton X-100 in antibody diluent for improved penetration

  • Extend washing steps to 4-6 × 10 minutes

• Co-localization with Neuronal Markers:

  • Combine with established neuronal markers (NeuN, MAP2, β-III-tubulin)

  • Include glial markers (GFAP, Iba1) to distinguish neuronal vs. glial expression

  • Consider tyramide signal amplification for detecting low-abundance targets

• Functional Correlation Approaches:

  • Correlate OR5H6 expression with electrophysiological recordings

  • Combine immunofluorescence with in situ hybridization for RNA-protein correlation

  • Implement activity-dependent markers to relate OR5H6 expression to neuronal function

These specialized approaches address the complexity of OR5H6 detection in neuronal tissues while maintaining specificity and reducing background interference characteristic of brain tissue samples.

How should I approach experimental design for studying OR5H6 interactions with other proteins?

A comprehensive experimental design for investigating OR5H6 protein interactions requires multi-technique validation:

• Co-immunoprecipitation Strategy:

  • Use unconjugated OR5H6 antibody for pull-down experiments

  • Verify protein interactions with reciprocal co-IP experiments

  • Include appropriate controls (IgG control, lysate input)

  • Confirm specificity using cells lacking OR5H6 expression

• Microscopy-Based Interaction Studies:

  • Apply proximity ligation assay (PLA) to detect protein interactions in situ

  • Perform FRET analysis using OR5H6 Antibody, FITC as donor fluorophore

  • Establish appropriate positive and negative controls for each technique

  • Quantify co-localization using Manders' coefficient and Pearson's correlation

• Biochemical Validation:

  • Confirm interactions using orthogonal methods (GST pull-down, yeast two-hybrid)

  • Employ deletion mutants to map interaction domains

  • Use cross-linking approaches to stabilize transient interactions

  • Verify functional relevance through activity assays

• Advanced Proteomic Approaches:

  • Consider BioID or APEX2 proximity labeling with OR5H6 fusion proteins

  • Implement quantitative mass spectrometry to identify interaction partners

  • Use SILAC or TMT labeling for comparative interaction studies

  • Validate key interactions through targeted proteomics approaches

This multi-faceted approach provides rigorous validation of protein interactions while minimizing false positives that can arise from single-technique studies.

What are the detailed specifications of OR5H6 Antibody, FITC conjugated?

These specifications highlight the antibody's suitability for human OR5H6 detection in immunofluorescence applications, with careful consideration required for storage and handling to maintain optimal reactivity.

How does OR5H6 Antibody, FITC conjugated compare with other fluorophore-conjugated antibodies?

This comparison demonstrates that OR5H6 Antibody, FITC conjugated offers simplified workflow and direct detection, but may have lower sensitivity compared to amplified detection systems. Selection should be based on experimental requirements for sensitivity, multiplexing needs, and available instrumentation.

What emerging applications could utilize OR5H6 Antibody, FITC conjugated in neuroscience research?

OR5H6 Antibody, FITC conjugated is positioned to contribute to several emerging neuroscience research areas:

• Single-cell Neuronal Profiling: Integration with single-cell RNA sequencing data to correlate OR5H6 protein expression with transcriptomic profiles in olfactory neurons. This approach can reveal heterogeneity in olfactory receptor expression and potential non-canonical functions in specific neuronal subpopulations.

• Neuronal Circuit Mapping: Combining with advanced tissue clearing techniques (CLARITY, iDISCO) and light-sheet microscopy to map OR5H6-expressing neurons within intact neural circuits. This whole-brain mapping approach could identify previously unrecognized connections between olfactory and other brain regions.

• Neurodevelopmental Studies: Tracking OR5H6 expression during critical developmental windows to understand the temporal dynamics of olfactory receptor expression. Correlation with axon guidance molecules may reveal mechanisms underlying the precise wiring of the olfactory system.

• Olfactory Dysfunction in Neurological Disorders: Investigating alterations in OR5H6 expression in models of neurodegenerative diseases where olfactory dysfunction is an early symptom (Parkinson's, Alzheimer's). This antibody could serve as a molecular tool to identify cellular mechanisms underlying disease-associated olfactory deficits.

• Multi-omics Integration: Correlation of protein-level data from OR5H6 immunofluorescence with epigenetic, transcriptomic, and metabolomic datasets to develop comprehensive models of olfactory receptor regulation and function in health and disease states.

These emerging applications highlight the potential for OR5H6 Antibody, FITC conjugated to contribute to multidisciplinary approaches in modern neuroscience research beyond traditional immunofluorescence applications.

How should researchers approach validation of OR5H6 Antibody, FITC conjugated for novel applications?

A comprehensive validation strategy for novel applications of OR5H6 Antibody, FITC conjugated should follow these methodical steps:

• Analytical Validation:

  • Perform epitope mapping to confirm binding to the intended region (aa 277-292)

  • Verify antibody specificity using knockout/knockdown models

  • Conduct peptide competition assays with synthetic OR5H6 peptide

  • Compare staining patterns with alternative OR5H6 antibodies targeting different epitopes

• Technical Validation:

  • Optimize protocol parameters systematically (fixation, permeabilization, antibody concentration)

  • Document reproducibility across multiple batches and experimental runs

  • Establish dynamic range and detection limits for quantitative applications

  • Compare results with orthogonal detection methods (RNA-based approaches, mass spectrometry)

• Biological Validation:

  • Verify expected expression patterns in positive control tissues

  • Confirm absence of signal in tissues known not to express OR5H6

  • Validate response to experimental manipulations known to alter OR5H6 expression

  • Correlate findings with functional readouts where applicable

• Application-Specific Validation:

  • For novel tissue types: establish tissue-specific optimization parameters

  • For new species: confirm epitope conservation through sequence alignment

  • For novel techniques: benchmark against established methods

  • For quantitative applications: demonstrate linearity, precision, and accuracy