Vertebrate ancient opsin Antibody, Biotin conjugated

What is Vertebrate ancient opsin and what role does it play in photosensitivity?

Vertebrate ancient (VA) opsin is a photoreceptive protein first isolated from Atlantic salmon and subsequently identified in various teleost fish and birds . It belongs to the opsin family of G-protein coupled receptors that mediate light sensitivity. VA opsin is expressed in multiple tissues including the retina, pineal gland, and sub-ependymal cells of the hypothalamus .

Unlike conventional visual opsins, VA opsin serves primarily in non-visual photoreception. Studies have revealed that VA opsin, particularly its variant VA-Long (VAL), forms functional photopigments with a maximum absorption (λmax) of approximately 460-500nm, making it a green-sensitive photopigment . These deep brain photoreceptors are implicated in regulating circadian rhythms, reproductive responses to photoperiod in birds, and potentially skin color regulation in fish dependent on environmental light conditions .

What are the key specifications of the Vertebrate ancient opsin Antibody, Biotin conjugated?

The Vertebrate ancient opsin Antibody, Biotin conjugated (product code: CSB-PA517517LD01SWI) is a polyclonal antibody raised in rabbits against Salmo salar (Atlantic salmon) Vertebrate ancient opsin . Its key specifications include:

This antibody is particularly suitable for research applications requiring high sensitivity detection systems that utilize biotin-avidin/streptavidin interactions .

What applications is the Vertebrate ancient opsin Antibody, Biotin conjugated suitable for?

The Vertebrate ancient opsin Antibody, Biotin conjugated has been tested and validated for ELISA applications as indicated in the product specifications . The biotin conjugation makes this antibody particularly suitable for detection systems employing avidin/streptavidin-based amplification.

While ELISA is the primary validated application, researchers may also consider using this antibody for other biotin-compatible techniques such as:

• Immunohistochemistry (IHC): For detecting VA opsin in fixed tissue sections, particularly in hypothalamic regions and retinal tissues where VA opsin is expressed .

• Immunocytochemistry (ICC): For cellular localization studies, especially when investigating VA opsin in cultured cells.

• Flow cytometry: When studying cell populations expressing VA opsin.

• Western blotting: Although not specifically validated, the antibody's dilution ratio for WB is suggested at 1:500-1:5000 , indicating potential compatibility.

For optimal results, preliminary validation experiments should be conducted when adapting this antibody to applications beyond ELISA.

What is the optimal storage and handling protocol for maintaining antibody activity?

To maintain maximum antibody activity and stability, the Vertebrate ancient opsin Antibody, Biotin conjugated should be stored according to the following recommendations:

• Upon receipt, store at -20°C or -80°C for long-term preservation .

• Avoid repeated freeze-thaw cycles, as these can significantly degrade antibody quality and reduce binding efficiency .

• For frequent use, small aliquots can be prepared and stored separately to minimize freeze-thaw cycles.

• When working with the antibody, thaw slowly on ice or at 4°C rather than at room temperature.

• Before use, centrifuge the antibody solution briefly to collect contents at the bottom of the tube.

• The antibody is supplied in a buffer containing 50% glycerol, which helps prevent freezing damage and maintains stability .

• Keep track of storage time and conditions, as antibody performance may decrease over extended storage periods.

Proper storage and handling are critical for maintaining the specificity and sensitivity of the antibody throughout your research project.

What methodological considerations should be addressed when designing immunohistochemistry experiments with this antibody in neural tissues?

When designing immunohistochemistry experiments using the Vertebrate ancient opsin Antibody, Biotin conjugated in neural tissues, several critical methodological considerations should be addressed:

• Fixation protocol: The choice between perfusion-fixation and immersion-fixation significantly impacts results. For deep brain tissues, perfusion with 2-4% paraformaldehyde is recommended, similar to protocols used for other opsin detection . Stronger fixatives containing glutaraldehyde (e.g., 2% paraformaldehyde/0.1% glutaraldehyde) may be used for electron microscopy studies but could reduce antigen accessibility .

• Antigen retrieval: For formalin-fixed, paraffin-embedded tissues, antigen retrieval methods such as trypsin treatment have proven effective for other opsin antibodies . This step might be essential for exposing epitopes masked during fixation, particularly for membrane proteins like opsins.

• Blocking protocol: Thorough blocking is crucial to minimize background, especially with biotin-conjugated antibodies. A blocking solution containing 3% serum (from the same species as the secondary antibody) with 0.1-0.4% Triton X-100 for permeabilization should be applied for 1-2 hours at room temperature .

• Biotin-streptavidin detection system: Since this antibody is biotin-conjugated, use a streptavidin-HRP or streptavidin-fluorophore conjugate. Be aware that endogenous biotin in brain tissues may cause background; consider using an avidin/biotin blocking kit before antibody application.

• Incubation conditions: For detecting low-abundance proteins like VA opsin in brain tissues, extend primary antibody incubation to 24-48 hours at 4°C with gentle agitation .

• Controls: Include appropriate negative controls (omitting primary antibody) and pre-absorption controls using the immunizing peptide when available to validate specificity .

• Counterstaining: Consider using DAPI for nuclear counterstaining to help identify specific cell populations and neuroanatomical structures.

• Section thickness: For brain tissues, 30-40μm sections are recommended for adequate penetration of antibodies while maintaining structural integrity.

• Signal amplification: For low-abundance targets, tyramide signal amplification can enhance detection sensitivity while maintaining specificity.

How does VA opsin differ functionally from other opsins, and what implications does this have for experimental design?

VA opsin exhibits several distinctive functional characteristics compared to other opsins, which necessitates specific experimental design considerations:

• Spectral sensitivity profiles: VA-Long (VAL) opsin forms a green-sensitive pigment with λmax ~500 nm when reconstituted with 11-cis-retinal, while standard VA opsin shows little to no photosensitivity in the same conditions . This spectral property differs from visual opsins like red/green (L/M) opsins and blue (S) opsins, which have distinct absorption maxima . When designing photoactivation experiments, researchers should use appropriate wavelengths centered around 500 nm for VA opsin stimulation.

• Cellular localization: Unlike typical visual opsins confined to photoreceptor outer segments, VA opsin is expressed in diverse tissues including retinal horizontal cells, pineal gland, and deep brain regions, particularly in cells surrounding the diencephalic ventricle . This diverse expression pattern requires careful tissue preparation and region-specific analysis protocols.

• Physiological roles: VA opsin mediates non-visual photosensory functions including circadian regulation and potentially skin color changes in response to environmental light in fish . These non-visual functions may require different physiological readouts (hormone levels, circadian gene expression, skin pigmentation changes) rather than standard visual function tests.

• Splicing variants: The existence of functionally distinct splicing variants (VA and VAL) means experiments must be designed to distinguish between these forms . Primers and antibodies must be selected with consideration for these variants.

• Retinal availability: VA opsin function depends on the presence of 11-cis-retinal as a chromophore . Experimental designs should account for the availability of this cofactor in model systems, which may vary between tissues.

• Co-localization with signaling partners: Unlike visual opsins with well-characterized phototransduction cascades, the downstream signaling pathways for VA opsin are less understood. Co-localization studies with potential signaling partners should be included in experimental designs.

• Temporal expression patterns: VA opsin expression may follow circadian rhythms or respond to seasonal photoperiod changes, particularly in birds . Experimental designs should control for time of day and seasonal effects.

These functional differences highlight the importance of tailored experimental approaches when studying VA opsin compared to conventional visual opsins.

What controls and validation methods are essential when characterizing new VA opsin-expressing tissues?

When characterizing new tissues for VA opsin expression, a comprehensive validation approach is essential to ensure specificity and reliability of findings:

• Antibody specificity controls:

  • Pre-absorption control: Incubate the antibody with excess immunizing peptide (recombinant Salmo salar VA opsin protein, 1-75AA) before application to tissue samples .

  • Western blot validation: Confirm antibody specificity by Western blot showing a single band at the expected molecular weight (~40 kDa for VA opsin) .

  • Knockout/knockdown controls: When available, tissues from VA opsin knockout or knockdown models provide definitive negative controls.

• Cross-species validation:

  • When studying VA opsin in species other than Atlantic salmon, validate antibody cross-reactivity using Western blot or dot blot analyses.

  • Consider using multiple antibodies targeting different epitopes of VA opsin if available.

• Multiple detection methods:

  • Corroborate protein detection with mRNA analysis (RT-PCR, in situ hybridization) to confirm expression at transcriptional level .

  • Quantitative PCR can validate expression levels across different tissues or conditions.

• Functional validation:

  • Demonstrate photosensitivity of identified cells using electrophysiological recordings or calcium imaging in response to 460-500 nm light stimulation .

  • Reconstitution experiments with expressed protein and 11-cis-retinal can confirm photopigment formation .

• Comparative anatomical analysis:

  • Compare the distribution pattern with known VA opsin-expressing tissues in related species.

  • Use neuroanatomical markers to precisely identify the location of positive cells within the tissue context.

• Co-localization studies:

  • Perform double-labeling with established cell-type specific markers to identify the cellular population expressing VA opsin.

  • For brain tissues, distinguish between neuronal and glial expression using appropriate markers .

• Technical controls:

  • Include no-primary-antibody controls to assess secondary antibody specificity.

  • For biotin-conjugated antibodies, include controls for endogenous biotin using avidin/biotin blocking systems.

  • Include positive control tissues with known VA opsin expression (e.g., specific regions of fish hypothalamus or retina) .

• Titration experiments:

  • Perform antibody dilution series to determine optimal concentration for specific signal versus background.

  • Compare staining patterns across different antibody concentrations to confirm specificity of detected signals.

How can researchers optimize double-labeling experiments involving Vertebrate ancient opsin Antibody, Biotin conjugated?

Double-labeling experiments combining Vertebrate ancient opsin Antibody, Biotin conjugated with other antibodies require careful optimization to ensure specific detection without cross-reactivity or interference. Here's a methodological approach:

• Sequential versus simultaneous incubation:

  • For biotin-conjugated VA opsin antibody, sequential detection is often preferred to minimize cross-reactivity.

  • Complete the detection of the non-biotinylated primary antibody first, followed by detection of the biotin-conjugated VA opsin antibody.

• Blocking endogenous biotin:

  • Before immunostaining, block endogenous biotin using an avidin/biotin blocking kit, especially critical in tissues like liver, kidney, and brain that contain high levels of endogenous biotin .

• Selection of compatible detection systems:

  • For the second primary antibody, choose one raised in a different host species than rabbit to avoid cross-reactivity.

  • Use fluorophore-labeled secondary antibodies with well-separated emission spectra for fluorescent detection:

    • For VA opsin detection: Streptavidin conjugated to a red fluorophore (e.g., Alexa Fluor 594)

    • For second target: Green fluorophore (e.g., Alexa Fluor 488) conjugated to appropriate secondary

• Optimized blocking protocol:

  • Use a blocking solution containing serum from the species of both secondary antibodies.

  • Include 0.3% Triton X-100 for membrane permeabilization with adequate blocking proteins (5% normal serum plus 1% BSA) .

• Cross-adsorbed secondary antibodies:

  • Use highly cross-adsorbed secondary antibodies to minimize species cross-reactivity.

• Control for bleed-through and cross-reactivity:

  • Run single-labeled controls with each primary antibody to ensure no bleed-through between channels.

  • Include negative controls omitting each primary antibody separately to check for non-specific binding.

• Optimization of signal amplification:

  • Titrate streptavidin-conjugate concentration to balance signal strength with background.

  • If signal amplification is needed, tyramide signal amplification can be employed for the biotin-conjugated antibody.

• Confocal microscopy settings:

  • Use sequential scanning rather than simultaneous scanning to eliminate channel cross-talk.

  • Adjust detector gain settings to minimize background while maintaining specific signal.

• Fixation considerations:

  • For double-labeling experiments in neural tissues, 4% paraformaldehyde fixation without glutaraldehyde is generally preferred to maintain epitope accessibility .

• Validation of co-localization:

  • Use appropriate co-localization analysis methods (Pearson's correlation, Manders' overlap coefficient) to quantify the degree of co-localization.

  • Confirm co-localization in multiple optical sections to rule out coincidental overlay.

What methodological approaches are recommended for quantifying VA opsin expression levels?

Quantifying VA opsin expression levels requires robust methodological approaches tailored to the specific experimental context. Here are recommended methods for accurate quantification:

• Western blot quantification:

  • Use standardized protein extraction protocols across samples.

  • Include calibration standards on each blot to create a standard curve.

  • Normalize VA opsin band intensity to housekeeping proteins (β-actin, GAPDH).

  • Use dilution series of 1:500-1:5000 as recommended for the antibody .

  • Employ image analysis software (ImageJ, Image Studio) for densitometry analysis.

  • Include positive controls from tissues known to express VA opsin.

• ELISA-based quantification:

  • Develop a sandwich ELISA using the biotin-conjugated antibody as the detection antibody.

  • Create a standard curve using recombinant VA opsin protein at known concentrations.

  • Use recommended dilutions of 1:2000-1:10000 for optimal sensitivity .

  • Subtract background signals from negative control samples.

  • Report results as absolute concentration based on standard curve interpolation.

• Immunohistochemical quantification:

  • Use consistent staining protocols across all experimental groups.

  • Employ stereological methods for unbiased cell counting:

    • Optical fractionator for estimating positive cell numbers

    • Cavalieri estimator for volume measurements

  • For fluorescence intensity measurement:

    • Capture images with identical exposure settings

    • Define region of interest (ROI) consistently across samples

    • Measure integrated density or mean gray value within ROIs

    • Subtract background measured from adjacent negative areas

• RT-qPCR for transcript quantification:

  • Design primers specific for VA opsin, differentiating between VA and VAL variants if necessary .

  • Use multiple reference genes validated for stability in your tissue of interest.

  • Apply the 2^(-ΔΔCt) method for relative quantification.

  • Include no-template and no-RT controls.

  • Validate PCR efficiency using standard curves.

• Single-cell analysis approaches:

  • For heterogeneous tissues like retina and brain, consider single-cell RNA-seq to identify specific cell populations expressing VA opsin.

  • Use fluorescence-activated cell sorting (FACS) combined with immunostaining to isolate and quantify VA opsin-positive cell populations.

• Biosensor measurements:

  • Consider using surface plasmon resonance (BIA core) for quantitative measurements of antibody-antigen interactions as described for other opsin antibodies .

  • Report binding strength in resonance units (RU) as a measure of antigen-antibody interaction.

• Normalization strategies:

  • Normalize to total protein content for biochemical assays.

  • For tissue sections, normalize to section thickness and area measured.

  • Account for regional variations in expression by sampling multiple areas.

• Statistical analysis:

  • Use appropriate statistical tests based on data distribution (parametric vs. non-parametric).

  • Account for multiple comparisons when analyzing various brain regions or experimental conditions.

  • Report effect sizes alongside p-values to indicate biological significance.

What are the technical challenges in detecting low-abundance VA opsin in deep brain photoreceptors and how can they be addressed?

Detecting low-abundance VA opsin in deep brain photoreceptors presents several technical challenges that require specialized approaches:

• Limited cellular distribution:

  • VA opsin-positive cells in brain tissues are often restricted to specific regions (e.g., cells surrounding the diencephalic ventricle, central thalamus) and may be sparsely distributed .

  • Solution: Use precise stereotaxic coordinates for tissue sampling and section at multiple levels through regions of interest to avoid missing areas of expression.

• Signal-to-noise ratio:

  • Low abundance proteins yield weak signals that can be difficult to distinguish from background.

  • Solution: Implement signal amplification techniques such as tyramide signal amplification (TSA), which can enhance detection sensitivity by 10-100 fold while maintaining specificity.

• Autofluorescence in brain tissue:

  • Neural tissues often exhibit high autofluorescence, particularly after fixation.

  • Solution: Employ autofluorescence reduction techniques such as Sudan Black B treatment (0.1-0.3%) or utilize spectral unmixing during confocal microscopy.

• Fixation-induced epitope masking:

  • Fixatives can mask epitopes, particularly for membrane proteins like opsins.

  • Solution: Optimize fixation protocols (shorter fixation times, lower fixative concentrations) and implement antigen retrieval methods. Heat-induced epitope retrieval in citrate buffer (pH 6.0) or enzymatic retrieval with trypsin has proven effective for other opsin antibodies .

• Limited antibody penetration:

  • Deep brain sections may suffer from inadequate antibody penetration.

  • Solution: Use thinner sections (30-40μm), extend incubation times (48-72 hours at 4°C), and include detergents (0.3-0.5% Triton X-100) to enhance penetration .

• Cross-reactivity with other opsins:

  • The brain expresses multiple opsin types that could potentially cross-react.

  • Solution: Validate antibody specificity through Western blot confirmation and pre-absorption controls. Consider parallel staining with antibodies against other opsins to confirm distinct populations.

• Availability of suitable controls:

  • Negative controls for deep brain photoreceptors can be challenging to establish.

  • Solution: Use tissues from species where VA opsin expression has not been reported as negative controls. When available, tissues from VA opsin knockout models provide definitive negative controls.

• Rostrocaudal distribution:

  • VA opsin-positive cells may be distributed over 200μm along the rostrocaudal axis , making them easy to miss in standard sections.

  • Solution: Serial sectioning and systematic sampling throughout the region of interest, with 3D reconstruction to visualize the complete distribution pattern.

• Biotin-related background in brain tissue:

  • Endogenous biotin in brain tissue can lead to false-positive signals when using biotin-conjugated antibodies.

  • Solution: Implement a specific endogenous biotin-blocking step using avidin/biotin blocking kits before applying the biotin-conjugated primary antibody.

• Co-localization with neuroanatomical markers:

  • Identifying precise neuroanatomical location of positive cells is crucial for functional interpretation.

  • Solution: Perform double-labeling with established neuroanatomical markers and validate using stereotaxic brain atlases specific to your species of interest.

• Circadian variation in expression:

  • VA opsin expression may exhibit circadian variation, complicating quantitative comparisons.

  • Solution: Perform tissue collection at consistent zeitgeber times across experimental groups and consider time-course studies to identify potential temporal patterns in expression.

By addressing these technical challenges with appropriate methodological adjustments, researchers can enhance the detection sensitivity and specificity of VA opsin in deep brain photoreceptors.