Identification of Orexin and Endocannabinoid Receptors in Adult Zebrafish Using Immunoperoxidase and Immunofluorescence Methods
Summary
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.
Abstract
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.
Introduction
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’-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.
Protocol
Representative Results
Discussion
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…
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
This study was supported by Fondi Ricerca di Ateneo (FRA)2015-2016, University of Sannio.
Materials
| Anti CB1 | Abcam | ab23703 | |
| Anti OX-2R | Santa Cruz | sc-8074 | |
| Anti-OXA | Santa Cruz | sc8070 | |
| Aquatex | Merck | 1,085,620,050 | |
| Biotinylated rabbit anti-goat | Vector Lab | BA-5000 | |
| citric acid | Sigma Aldrich | 251275 | |
| Confocal microscope | Nikon | Nikon Eclipse Ti2 | |
| Cryostat | Leica Biosystem | CM3050S | |
| DAPI | Sigma Aldrich | 32670 | |
| Digital Camera | Leica Biosystem | DFC320 | |
| Digital Camera for confocal microscope | Nikon | DS-Qi2 | |
| Donkey anti goat Alexa fluor 488-conjugated secondary antibodies | Thermo Fisher | A11055 | |
| Donkey anti goat Alexa fluor 594-conjugated secondary antibodies | Thermo Fisher | A11058 | |
| Donkey anti rabbit Alexa fluor 488-conjugated secondary antibodies | Thermo Fisher | A21206 | |
| Donkey anti rabbit Alexa fluor 594-conjugated secondary antibodies | Thermo Fisher | A21207 | |
| Ethanol absolute | VWR | 20,821,330 | |
| Frozen section compound | Leica Biosystem | FSC 22 Frozen Section Media | |
| H2O2 | Sigma Aldrich | 31642 | |
| HCl | VWR | 20,252,290 | |
| ImmPACT DAB | Vector lab | SK4105 | |
| Microscope | Leica Biosystem | DMI6000 | |
| Microtome | Leica Biosystem | RM2125RT | |
| Na2HPO4 | Sigma Aldrich | S9763 | |
| NaCl | Sigma Aldrich | S7653 | |
| NaH2PO4H2O | Sigma Aldrich | S9638 | |
| NaOH | Sigma Aldrich | S8045 | |
| Normal Donkey Serum | Sigma Aldrich | D9663 | |
| Normal Rabbit Serum | Vector lab | S-5000 | |
| paraffin wax | Carlo Erba | 46793801 | |
| Paraphormaldeyde | Sigma Aldrich | P6148 | |
| sodium citrate dihydrate | Sigma Aldrich | W302600 | |
| Triton X-100 | Fluka Analytical | 93420 | |
| Trizma | Sigma Aldrich | T1503 | |
| VectaStain Elite ABC Kit | Vector lab | PK6100 | |
| Xylene Pure | Carlo Erba | 392603 |
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