Use of zebrafish for cardiovascular research is expanding towards research on adult hearts. For these applications, quick and simple isolation of cardiac tissues is key to avoid post-mortem changes and to obtain an adequate number of samples. Here, we describe a fast and reproducible method for dissecting adult zebrafish hearts.
Bruk av sebrafisk modellsystem for å studere utviklingen, regenerering, og sykdom ekspanderer mot bruk av voksne hjerter for celledissosiasjon og rensing av RNA, DNA og proteiner. Alle disse programmene krever rask gjenoppretting av et betydelig antall av sebrafisk hjerter for å unngå gen regulatoriske, metabolsk, og andre endringer som begynner etter døden. Voksne sebrafisk hjerter er også nødvendig for å studere hjertestruktur for en rekke mutanter og for å studere hjerte regenerering. Imidlertid er den tradisjonelle sebrafisk hjerte disseksjon langsom og vanskelig og krever spesialverktøy, slik at storskala disseksjon av voksne sebrafisk hjerter langtekkelig. Tradisjonelle metoder også havn risikoen for å skade hjertet under disseksjon. Her beskriver vi en metode for disseksjon av voksne sebrafisk hjerter som er rask, reproduserbar, og bevarer hjerte arkitektur. Videre gjør denne metoden ikke krever spesialverktøy, er smertefri for sebrafisk,kan utføres på friske eller faste prøver, og kan utføres på sebrafisk som ung som en måned gammel. Tilnærmingen beskrevet utvider bruken av voksen sebrafisk for kardiovaskulær forskning.
Zebrafish are an excellent model for studying heart development and human disease1,2. Specific advantages include the translucent nature of zebrafish embryos, the availability of many genetic mutants and transgenic reporter lines, and the availability of genome editing technologies. In addition to their advantages for studying early heart development, zebrafish are an ideal system for studying vertebrate heart regeneration3.
More recently, adult zebrafish are playing an important part in bioinformatics approaches to studying cardiovascular development and disease, due to their relatively large clutch size and relatively quick and inexpensive breeding compared to other vertebrate models. Promising techniques include ribosome profiling, RNA-Seq, and cell dissociation and FACS sorting4-7. However, for these techniques the quality of the data can depend on obtaining a large number of samples in a rapid, efficient, and reproducible manner, before gene regulatory, metabolic, transcriptional, and other changes occur.
Dissection of adult zebrafish organs has been described in the past8,9. However, previous approaches to dissection of the heart were slow, ran the risk of damaging the heart during dissection, required special tools, and/or required fixation of the zebrafish prior to dissection; for these reasons, past approaches to zebrafish adult heart dissection were not optimized for high-throughput applications and/or applications requiring fresh tissue.
Here, we describe a method for adult zebrafish heart dissection that is simple, fast, efficient, and reproducible, while preserving cardiac morphology. This method does not include cutting into the pericardial space and therefore does not risk damaging the heart during dissection. Instead, this method relies on anatomical landmarks of the zebrafish, and therefore, it is highly reproducible. This dissection method is also versatile in that it can be used on fresh or fixed fish, and on zebrafish as young as one month old. Finally, this method results in minimal suffering to the zebrafish because after anesthesia and/or rapid cooling, the fish is additionally decapitated and pithed in the course of the dissection procedure.
Selv om fremgangsmåter for å dissekere den voksne sebrafisk hjertet er blitt beskrevet, er disse metoder var tidkrevende og ofte forårsaket skade på hjertet under disseksjonen. For å utføre eksperimenter hvor et stort antall voksne hjerter kan være nødvendig, og / eller ved å unngå nedbrytning av hjertevevet er viktig for nedstrøms applikasjoner, er prohibitive den tid som kreves ved bruk av tradisjonelle teknikker disseksjon. Tilsvar, reproduserbart skaffe uskadde, intakte hjerter er viktig for studier av hj…
The authors have nothing to disclose.
The authors would like to thank Dr. Shaun Coughlin for hosting the filming of this procedure in his laboratory, and for general support. R.A. was supported by the NIH (F32HL110489) and the Sarnoff Cardiovascular Research Foundation. S.R. was supported by a Research Fellowship of the Deutsche Forschungsgemeinschaft (DFG) and the American Heart Association (AHA). D.Y.R.S was supported by the NIH (RO1HL54737), the Packard Foundation, and the Max Planck Society.
small tank for transporting fish | Aquaneering | ZHCT100 | |
fish net | Petsmart | 36-16731 | |
250mL glass beaker | Kimble | 14005-250 | |
9cm polystyrene petri dish | Nunc | 172958 | |
razor blade | Personna American Safety Razor Company | 94-120-71 | |
two Dumont #5SF forceps | Fine Science Tools | 11252-00 | |
dissecting microscope | Olympus | SZX16 | |
Tricaine | Sigma | A-5040 | |
plastic transfer pipette | Thermo Scientific | 202-20S | |
gooseneck light source | Dolan-Jenner Industries, Inc | Fiber-Lite 180 Illuminator, 181 Dual Gooseneck System | |
fluorescent light source | Lumen Dynamics | X-Cite 120Q | optional |
micro-scissors | Biomedical Research Instruments, Inc | 11-1000 | optional |
RBC Lysis Buffer | eBioscience | 00-4333-57 | optional |