This study was supported by Fondi Ricerca di Ateneo (FRA)2015-2016, University of Sannio.

The authors declare no conflicts of interest.

Sample preparation
Sample preparation is the first critical step in IHC. A reliable protocol allows for maintenance of cell morphology, tissue architecture, and antigenicity. This step requires correct tissue collection, fixation, and sectioning22,23. The purpose of fixation is to preserve tissue and reduce the action of tissue enzymes or microorganisms. In particular, the fixation step preserves cellular compon...

Immunohistochemistry (IHC) is a well-established classic technique used to identify cellular or tissue components (antigens) by antigen-antibody interaction1,2. It can be used to identify the localization and distribution of target biomolecules within a tissue. IHC uses immunological and chemical reactions to detect antigens in tissue sections3. The main markers used for the visualization of antigen-antibody interactions include fluorescent dyes (immunofluorescence) and enzyme-substrate color reactions (immunoperoxidase), both conjugated to antibodies4. Using microscopic observation is possible to determine the localization of labeled tissue, which approximately corresponds to localization of the target antigen in the tissue.
Two methods exist for fluorescent or chromogenic reactions to detect protein: the direct detection method, in which the specific primary antibody is directly labeled; and the indirect detection method, in which the primary antibody is unconjugated while the secondary antibody carries the label5,6,7. The indirect method has some advantages, which is mainly its signal amplification. Moreover, unlike other molecular and cellular techniques, with immunofluorescence, it is possible to visualize the distribution, localization, and coexpression of two or more proteins differentially expressed within cells and tissues7. The choice of the detection method used depends on experimental details.
To date, IHC is widely used in basic research as a powerful and essential tool to understanding the distribution and localization of biomarkers and the general profiling of different proteins in biological tissue from human to invertebrates8,9,10,11. The technique helps display a map of protein expression in a large number of normal and altered animal organs and different tissue types, showing possible down- or up-regulation of expression induced by physiological and pathological changes. IHC is a highly sensitive technique that requires accuracy and the correct choice of methods to obtain optimal results12. First of all, many different factors such as fixation, cross-reactivity, antigen retrieval, and sensitivity of antibodies can lead to false positive and false negative signals13. Selection of the antibodies is one of the most important steps in IHC and depends on the antigen specificity and its affinity to the protein and species under investigation7.
Recently, we have optimized the IHC technique to detect members of orexin/hypocretin and endocannabinoid systems in adult zebrafish tissue. We have focused mainly on fixation, tissue embedding using two different approaches, sectioning and mounting (which can affect resolution and detail during microscopic analysis), and blocking (to prevent false positives and reduce background)14. Other important characteristics are the antibody specificity and selectivity and reproducibility of individual IHC protocols. The key to providing antibody specificity is the use of negative controls (including no primary antibodies or tissue that is known to not express the target proteins) as well as positive controls (including tissue that is known to express the target proteins)15. The selection of antibodies for IHC is made based on their species-specificity (the likelihood with which they react with the antigen of interest) and the antigen-antibody binding detection systems that is used4,5,6,7. In the case of immunoperoxidase, the color of the reaction is determined by selection of the precipitating chromogen, usually diaminobenzidine (brown)16. On the other hand, immunofluorescence utilizes antibodies conjugated with a fluorophor to visualize protein expression in frozen tissue sections and allows for easy analysis of multiple proteins with respect to the chromogenic detection system5,7.
In the immunoperoxidase technique, the secondary antibody is conjugated to biotin, a linker molecule capable of recruiting a chromogenic reporter molecule [avidin-biotin complex (ABC)], leading to amplification of the staining signal. With the ABC reporter method, the enzyme peroxidase reacts with 3,3&#8217;-diaminobenzidine (DAB), producing an intensely brown-colored staining where the enzyme binds to the secondary antibody, which can then be analyzed with an ordinary light microscope. ABC staining, due to the high affinity of avidin for biotin, produces a rapid and optimal reaction, with few secondary antibodies attached to the site of the primary antibody reactivity. This chromogenic detection method allows for the densitometric analysis of the signal, providing semi-quantitative data based on the correlation of brown signal levels with protein expression levels18.
With immunofluorescence techniques, simultaneous detection of multiple proteins is possible due to the ability of different fluorochromes to emit light at unique wavelengths, but is important to choose fluorochromes carefully to minimize spectral overlap5. Moreover, the use of primary antibodies in different host species minimizes difficulties concerning cross-reactivity. In this case, each species-specific secondary antibody recognizes only one type of primary antibody. Fluorescent reporters are small organic molecules, including commercial derivatives, such as Alexa Fluor dyes.
Many animal models are used to understand particular physiological and pathological conditions. To date, it is established that many metabolic pathways are conserved over the course of evolution. Therefore, IHC studies in model organisms such as zebrafish can provide insight into the genesis and maintenance of pathological and non-pathological conditions17. It is an aim of this report to illustrate IHC protocols that can be performed on adult zebrafish tissue and used to obtain detailed images of the distribution and localization of OXA, OX-2R, and CB1R at peripheral and central levels. Also reported are protocols for the application of two major IHC indirect methods in peripheral and central tissues of adult zebrafish. Described is the indirect method, which allows for signal amplification in cases where a secondary antibody is conjugated to a fluorescent dye (immunofluorescence method) or enzyme reporter (immunoperoxidase method). Both chromogenic and fluorescent detection methods possess advantages and disadvantages. Reported in this protocol is the use of IHC, mainly immunofluorescence, in adult zebrafish, an animal model widely used to study systems that are evolutionary conserved across different physiological and pathological conditions.

<table><tbody><tr><td>Anti CB1</td><td>Abcam</td><td>ab23703</td><td></td></tr><tr><td>Anti OX-2R</td><td>Santa Cruz</td><td>sc-8074</td><td></td></tr><tr><td>Anti-OXA</td><td>Santa Cruz</td><td>sc8070</td><td></td></tr><tr><td>Aquatex</td><td>Merck</td><td>1,085,620,050</td><td></td></tr><tr><td>Biotinylated rabbit anti-goat</td><td>Vector Lab</td><td>BA-5000</td><td></td></tr><tr><td>citric acid</td><td>Sigma Aldrich</td><td>251275</td><td></td></tr><tr><td>Confocal microscope</td><td>Nikon</td><td>Nikon Eclipse Ti2</td><td></td></tr><tr><td>Cryostat</td><td>Leica Biosystem</td><td>CM3050S</td><td></td></tr><tr><td>DAPI</td><td>Sigma Aldrich</td><td>32670</td><td></td></tr><tr><td>Digital Camera</td><td>Leica Biosystem</td><td>DFC320</td><td></td></tr><tr><td>Digital Camera for confocal microscope</td><td>Nikon</td><td>DS-Qi2</td><td></td></tr><tr><td>Donkey anti goat Alexa fluor 488-conjugated secondary antibodies</td><td>Thermo Fisher</td><td>A11055</td><td></td></tr><tr><td>Donkey anti goat Alexa fluor 594-conjugated secondary antibodies</td><td>Thermo Fisher</td><td>A11058</td><td></td></tr><tr><td>Donkey anti rabbit Alexa fluor 488-conjugated secondary antibodies</td><td>Thermo Fisher</td><td>A21206</td><td></td></tr><tr><td>Donkey anti rabbit Alexa fluor 594-conjugated secondary antibodies</td><td>Thermo Fisher</td><td>A21207</td><td></td></tr><tr><td>Ethanol absolute</td><td>VWR</td><td>20,821,330</td><td></td></tr><tr><td>Frozen section compound</td><td>Leica Biosystem</td><td>FSC 22 Frozen Section Media</td><td></td></tr><tr><td>H2O2</td><td>Sigma Aldrich</td><td>31642</td><td></td></tr><tr><td>HCl</td><td>VWR</td><td>20,252,290</td><td></td></tr><tr><td>ImmPACT DAB</td><td>Vector lab</td><td>SK4105</td><td></td></tr><tr><td>Microscope</td><td>Leica Biosystem</td><td>DMI6000</td><td></td></tr><tr><td>Microtome</td><td>Leica Biosystem</td><td>RM2125RT</td><td></td></tr><tr><td>Na2HPO4</td><td>Sigma Aldrich</td><td>S9763</td><td></td></tr><tr><td>NaCl</td><td>Sigma Aldrich</td><td>S7653</td><td></td></tr><tr><td>NaH2PO4H2O</td><td>Sigma Aldrich</td><td>S9638</td><td></td></tr><tr><td>NaOH</td><td>Sigma Aldrich</td><td>S8045</td><td></td></tr><tr><td>Normal Donkey Serum</td><td>Sigma Aldrich</td><td>D9663</td><td></td></tr><tr><td>Normal Rabbit Serum</td><td>Vector lab</td><td>S-5000</td><td></td></tr><tr><td>paraffin wax</td><td>Carlo Erba</td><td>46793801</td><td></td></tr><tr><td>Paraphormaldeyde</td><td>Sigma Aldrich</td><td>P6148</td><td></td></tr><tr><td>sodium citrate dihydrate</td><td>Sigma Aldrich</td><td>W302600</td><td></td></tr><tr><td>Triton X-100</td><td>Fluka Analytical</td><td>93420</td><td></td></tr><tr><td>Trizma</td><td>Sigma Aldrich</td><td>T1503</td><td></td></tr><tr><td>VectaStain Elite ABC Kit</td><td>Vector lab</td><td>PK6100</td><td></td></tr><tr><td>Xylene Pure</td><td>Carlo Erba</td><td>392603</td><td></td></tr></tbody></table>

identification of orexin and endocannabinoid receptors in adult zebrafish using immunoperoxidase and immunofluorescence methods

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled antibodies. It is a multistep process in which the optimization of each step is crucial to obtain the optimum specific signal. Through IHC, the distribution and localization of specific biomarkers can be detected, revealing information on evolutionary conservation. Moreover, IHC allows for the understanding of expression and distribution changes of biomarkers in pathological conditions, such as obesity. IHC, mainly the immunofluorescence technique, can be used in adult zebrafish to detect the organization and distribution of phylogenetically conserved molecules, but a standard IHC protocol is not estasblished. Orexin and endocannabinoid are two highly conserved systems involved in the control of food intake and obesity pathology. Reported here are protocols used to obtain information about orexin peptide (OXA), orexin receptor (OX-2R), and cannabinoid receptor (CB1R) localization and distribution in the gut and brain of normal and diet-induced obese (DIO) adult zebrafish models. Also described are methods for immunoperoxidase and double immunofluorescence, as well as preparation of reagents, fixation, paraffin-embedding, and cryoprotection of zebrafish tissue and preparation for an endogenous activity-blocking step and background counterstaining. The complete set of parameters is obtained from previous IHC experiments, through which we have shown how immunofluorescence can help with the understanding of OXs, OX-2R, and CB1R distribution, localization, and conservation of expression in adult zebrafish tissues. The resulting images with highly specific signal intensity led to the confirmation that zebrafish are suitable animal models for immunohistochemical studies of distribution, localization, and evolutionary conservation of specific biomarkers in physiological and pathological conditions. The protocols presented here are recommended for IHC experiments in adult zebrafish.

Representative data for the immunoperoxidase staining are shown in Figure 1 and Figure 2. Immunohistochemical analysis of OX-A and OX-2R distribution in the gut of adult zebrafish showed different localization sites of OX-A and OX-2R and their increases in expression in the intestinal cells of DIO zebrafish. An intense brown staining for OX-A was observed in the cells of the medial and anterior intestine (Figure 1A, A1). ...

Watch this Scientific Journal Video about Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods at JoVE.com

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Presented here are protocols for immunohistochemical characterization and localization of orexin peptide, orexin receptors, and endocannabinoid receptors in the gut and brains of normal and diet-induced obesity (DIO) adult zebrafish models using immunoperixidase and double immunofluorescence methods.

Immunohistochemistry (IHC) is a highly sensitive and specific technique involved in the detection of target antigens in tissue sections with labeled ...

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Research

JoVE Journal

Biochemistry

7.9K Views.  University of Sannio. Momenteel wordt immunohistochemie steeds vaker gebruikt om de aanwezigheid van specifieke moleculaire markers en hun veranderingen in het geval van pathologieën te identificeren, waardoor de mogelijkheid om kwantitatieve analyse uit te voeren, wortel geeft. Immunohistochemie, voornamelijk de immunofluorescentietechniek, wordt gebruikt in de larve en het volwassen zebravisdiermodel, maar er is weinig bekend over een goed standaard immunohistochemieprotocol voor zebravissen. Hier beschrijven e...

Video: Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

Developmental Biology

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities 

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the visual scene. However, the OKR requires that an animal detect moving stripes and it is possible that fish that fail to exhibit an OKR may not be completely blind. One zebrafish mutant, the no optokinetic response c (nrc) has no OKR under any light conditions tested and was reported to be completely blind. Previously, we have shown that OFF-ganglion cell activity can be recorded in these mutants. To determine whether mutant fish with no OKR such as the nrc mutant can detect simple light increments and decrements we developed the visual motor behavioral assay (VMR). In this assay, single zebrafish larvae are placed in each well of a 96-well plate allowing the simultaneous monitoring of larvae using an automated video-tracking system. The locomotor responses of each larva to 30 minutes light ON and 30 minutes light OFF were recorded and quantified. WT fish have a brief spike of motor activity upon lights ON, known as the startle response, followed by return to lower-than baseline activity, called a freeze. WT fish also sharply increase their locomotor activity immediately following lights OFF and only gradually (over several minutes) return to baseline locomotor activity. The nrc mutants respond similarly to light OFF as WT fish, but exhibit a slight reduction in their average activity as compared to WT fish. Motor activity in response to light ON in nrc mutants is delayed and sluggish. There is a slow rise time of the nrc mutant response to light ON as compared to WT light ON response. The results indicate that nrc fish are not completely blind. Because teleosts can detect light through non-retinal tissues, we confirmed that the immediate behavioral responses to light-intensity changes require intact eyes by using the chokh (chk) mutants, which completely lack eyes from the earliest stages of development. In our VMR assay, the chk mutants exhibit no startle response to either light ON or OFF, showing that the lateral eyes mediate this behavior. The VMR assay described here complements the well-established OKR assay, which does not test the ability of zebrafish larvae to respond to changes in light intensities. Additionally, the automation of the VMR assay lends itself to high-throughput screening for defects in light-intensity driven visual responses.

A Behavioral Assay to Measure Responsiveness of Zebrafish to Changes in Light Intensities

We developed the Visual-Motor Response to quantitate the motor output of larval zebrafish in response to light increments and decrements. We also examined zebrafish vision mutants, including the no optokinetic response (nrc) mutants, which were thought to be completely blind when tested by another vision assay, the optokinetic reflex.

The optokinetic reflex (OKR) is a basic visual reflex exhibited by most vertebrates and plays an important role in stabilizing the eye relative to the ...

Biology

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Electrophysiological Recording in the Brain of Intact Adult Zebrafish

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram recordings. This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments, allowing recording of neural activity. Immobilization of the adult requires a mechanism to deliver dissolved oxygen to the gills in lieu of buccal and opercular movement. With our technique, animals are immobilized and perfused with habitat water to fulfill this requirement. A craniotomy is performed under tricaine methanesulfonate (MS-222; tricaine) anesthesia to provide access to the brain. The primary electrode is then positioned within the craniotomy window to record extracellular brain activity. Through the use of a multitube perfusion system, a variety of pharmacological compounds can be administered to the adult fish and any alterations in the neural activity can be observed. The methodology not only allows for observations to be made regarding changes in neurological activity, but it also allows for comparisons to be made between larval and adult zebrafish. This gives researchers the ability to identify the alterations in neurological activity due to the introduction of various compounds at different life stages.

This paper describes how an adult zebrafish can be immobilized, intubated, and used for in vivo electrophysiological experiments to allow recordings and manipulation of neural activity in an intact animal.

Previously, electrophysiological studies in adult zebrafish have been limited to slice preparations or to eye cup preparations and electrorentinogram ...

Neuroscience

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a model for human diseases. Gene function study is based on the detection of gene expression patterns. Although immunohistochemistry offers a powerful way to assay protein expression, the limited number of commercially available antibodies in zebrafish restricts the application of costaining. In situ hybridization is widely used in zebrafish embryos to detect mRNA expression. This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry for zebrafish embryo sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. Immunohistochemistry and the imaging of a single cryosection were performed after in situ hybridization. The protocol is helpful to unravel the expression pattern of two genes, first by in situ transcript detection and then by immunohistochemistry against a protein in the same section.

In Situ Hybridization Combined with Immunohistochemistry in Cryosectioned Zebrafish Embryos

This protocol describes how to obtain images by combining in situ hybridization and immunohistochemistry of zebrafish embryonic sections. In situ hybridization was performed prior to cryosectioning, followed by antibody staining. It is useful to detect the expression patterns of two genes in zebrafish if there is a paucity of antibodies.

As a vertebrate, the zebrafish has been widely used in biological studies. Zebrafish and humans share high genetic homology, which allows its use as a ...

In Vivo Confocal Fluorescence Imaging of Neural Activity Induced by Sensory Stimulation in Partially Restrained Larval Zebrafish

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural circuitry facilitate the use of optogenetic techniques in cellular analyses of behavior. Fluorescent indicators of in vivo neural activity, such as GCaMP6s, have been widely used to study the neural activity associated with simple behaviors in larval zebrafish. Here, we present a protocol for detecting sensory-induced activity in semi-restrained zebrafish larvae using the transgenic line Tg(elav3:GCaMP6s). In particular, we use the chemical agent allyl isothiocyanate to induce a robust, reproducible fluorescent response in a brain region at the border of the hindbrain and spinal cord. We discuss the potential uses of GCaMP6s for optical monitoring of neural activity during a range of behavioral paradigms and the limitations of this technique. Our protocol outlines an accessible approach for monitoring dynamic, behavior-related in vivo neural activity in the larval zebrafish brain.

Here, we present a detailed protocol to examine neural activity in brain regions of transgenic zebrafish that express GCaMP calcium indicators using confocal microscopy.

Zebrafish larvae are a promising vertebrate model system for studying the neural mechanisms of behavior. Their translucence and relatively simple neural ...

Monitoring On-Target Signaling Responses in Larval Zebrafish - Z-REX Unmasks Precise Mechanisms of Electrophilic Drugs and Metabolites

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the mode of action (MOA) of such molecules leverage bulk treatment of experimental specimens with an excess of a specific reactive species. In this approach, the high reactivity of electrophiles renders non-discriminate labeling of the proteome in a time- and context-dependent manner; redox-sensitive proteins and processes can also be indirectly and often irreversibly affected. Against such a backdrop of innumerable potential targets and indirect secondary effects, linking phenotype to specific target engagement remains a complex task. Zebrafish targeting reactive electrophiles and oxidants (Z-REX)-an on-demand reactive-electrophile delivery platform adapted for use in larval zebrafish-is designed to deliver electrophiles to a specific protein of interest (POI) in otherwise unperturbed live fish embryos. Key features of this technique include a low level of invasiveness, along with dosage-, chemotype-, and spatiotemporally-controlled precision electrophile delivery. Thus, in conjunction with a unique suite of controls, this technique sidesteps off-target effects and systemic toxicity, otherwise observed following uncontrolled bulk exposure of animals to reactive electrophiles and pleiotropic electrophilic drugs. Leveraging Z-REX, researchers can establish a foothold in the understanding of how individual stress responses and signaling outputs are altered as a result of specific reactive ligand engagement with a specific POI, under near-physiologic conditions in intact living animals.

Zebrafish targeting reactive electrophiles and oxidants (Z-REX) is a chemical biology-based method for the investigation of reactive small-molecule signaling. This technique can be applied to live fish of different developmental stages. Here, we couple standard assays in zebrafish with Z-REX for signaling pathway analysis.

Reactive metabolites and related electrophilic drugs are among the most challenging small molecules to study. Conventional approaches to deconstruct the ...

Zebrafish Optogenetics: Activating Genetically Modified Somatosensory Neuron to Study Larval Behavioral Responses

Source: Palanca A. M. S., et al. <a href="https://www.jove.com/v/50184/" target="_blank">Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato</a>. J. Vis. Exp. (2013).
This video describes optogenetic techniques by activating genetically modified somatosensory neurons and recording elicited behavioral response with a high-speed video camera.

Source: Palanca A. M. S., et al. Optogenetic Activation of Zebrafish Somatosensory Neurons using ChEF-tdTomato. J. Vis. Exp. (2013).
This video describes ...

Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods

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