Name: recovery of adult zebrafish hearts for high-throughput applications
Uploaded: 2014-12-12T15:00:00
Description: Watch this Scientific Journal Video about Recovery of Adult Zebrafish Hearts for High-throughput Applications at JoVE.com

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.

NOTE: Always be sure that IACUC or ethics committee approval is in place before beginning any experimental procedure using zebrafish.
1. Prepare reagents and setup
<ol>
	<li>Prepare the following solutions. Find the recipes for all of these solutions in standard zebrafish manuals10.
	<ul>
		<li>500 ml of Egg Water</li>
		<li>200 ml of 0.03% Tricaine in Egg Water</li>
		<li>100 ml of 1x Phosphate-Buffered Saline (PBS)</li>
		<li>RBC Lysis Buffer (optional)</li>
		<li>Destination bu...

<ol>
	<li>Arnaout, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+model+for+human+long+QT+syndrome.">Zebrafish model for human long QT syndrome.</a> Proceedings of the National Academy of Sciences of the United States of America. 104 (27), 11316-11321 (2007).</li><li>Chi, N. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+physiologic+dissection+of+the+vertebrate+cardiac+conduction+system.">Genetic and physiologic dissection of the vertebrate cardiac conduction system.</a> PLoS Biology. 6 (5), 109 (2008).</li><li>Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Getting+to+the+heart+of+regeneration+in+zebrafish.">Getting to the heart of regeneration in zebrafish.</a> Seminars in Cell &amp; Developmental Biology. 18 (1), 36-45 (2007).</li><li>Fang, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translational+profiling+of+cardiomyocytes+identifies+an+early+Jak1/Stat3+injury+response+required+for+zebrafish+heart+regeneration.">Translational profiling of cardiomyocytes identifies an early Jak1/Stat3 injury response required for zebrafish heart regeneration.</a> Proceedings of the National Academy of Sciences of the United States of America. 110 (33), 13416-13421 (2013).</li><li>Manoli, M., Driever, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-activated+cell+sorting+(FACS)+of+fluorescently+tagged+cells+from+zebrafish+larvae+for+RNA+isolation.">Fluorescence-activated cell sorting (FACS) of fluorescently tagged cells from zebrafish larvae for RNA isolation.</a> Cold Spring Harbor Protocols. 2012 (8), (2012).</li><li>Cannon, J. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analysis+of+the+haematopoietic+and+endothelial+transcriptome+during+zebrafish+development.">Global analysis of the haematopoietic and endothelial transcriptome during zebrafish development.</a> Mechanisms of Development. 130 (2-3), 122-131 (2013).</li><li>Wang, Z., Gerstein, M., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+revolutionary+tool+for+transcriptomics.">RNA-Seq: a revolutionary tool for transcriptomics.</a> Nature Reviews Genetics. 10 (1), 57-63 (2009).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+organs+from+the+adult+zebrafish.">Dissection of organs from the adult zebrafish.</a> Journal of Visualized Experiments. 37, (2010).</li><li>Singleman, C., Holtzman, N. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+dissection+in+larval,+juvenile+and+adult+zebrafish,+Danio+rerio.">Heart dissection in larval, juvenile and adult zebrafish, Danio rerio.</a> Journal of Visualized Experiments. 55, (2011).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book: a guide for the laboratory use of zebrafish (Brachydanio rerio. , (1993).</li></ol>

While methods for dissecting the adult zebrafish heart have been described, these methods were time-consuming and commonly caused damage to the heart during dissection. To perform experiments where a large number of adult hearts may be needed, and/or when avoiding degradation of heart tissue is important for downstream applications, the time required using traditional dissection techniques is prohibitive. Similarly, reproducibly obtaining undamaged, intact hearts is important for study of heart structure and for immunohi...

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.

<table border="0" cellpadding="0" cellspacing="0" >
	<tbody>
		<tr >
			<td >small tank for transporting fish</td>
			<td >Aquaneering</td>
			<td >ZHCT100</td>
			<td></td>
		</tr>
		<tr >
			<td >fish net</td>
			<td >Petsmart</td>
			<td >36-16731</td>
			<td></td>
		</tr>
		<tr >
			<td >250mL glass beaker</td>
			<td >Kimble</td>
			<td >14005-250</td>
			<td></td>
		</tr>
		<tr >
			<td >9cm polystyrene petri dish</td>
			<td >Nunc</td>
			<td >172958</td>
			<td></td>
		</tr>
		<tr >
			<td >razor blade</td>
			<td >Personna American Safety Razor Company</td>
			<td >94-120-71</td>
			<td></td>
		</tr>
		<tr >
			<td >two Dumont #5SF forceps</td>
			<td >Fine Science Tools</td>
			<td >11252-00</td>
			<td></td>
		</tr>
		<tr >
			<td >dissecting microscope</td>
			<td >Olympus</td>
			<td >SZX16</td>
			<td></td>
		</tr>
		<tr >
			<td >Tricaine</td>
			<td >Sigma</td>
			<td >A-5040</td>
			<td></td>
		</tr>
		<tr >
			<td >plastic transfer pipette</td>
			<td >Thermo Scientific</td>
			<td >202-20S</td>
			<td></td>
		</tr>
		<tr >
			<td >gooseneck light source</td>
			<td >Dolan-Jenner Industries, Inc</td>
			<td >Fiber-Lite 180 Illuminator, 181 Dual Gooseneck System</td>
			<td></td>
		</tr>
		<tr >
			<td >fluorescent light source</td>
			<td >Lumen Dynamics</td>
			<td >X-Cite 120Q</td>
			<td>optional</td>
		</tr>
		<tr >
			<td >micro-scissors</td>
			<td >Biomedical Research Instruments, Inc</td>
			<td >11-1000</td>
			<td>optional</td>
		</tr>
		<tr >
			<td >RBC Lysis Buffer</td>
			<td >eBioscience</td>
			<td >00-4333-57</td>
			<td>optional</td>
		</tr>
	</tbody>
</table>

recovery of adult zebrafish hearts for high-throughput applications

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Using this method, an adult zebrafish heart can be dissected in less than 1 min, compared to over 5 min using traditional methods8. Hearts dissected using this method are reliably intact (Figure 2A), while traditional methods8 require cutting blindly into the pericardium and therefore commonly cause damage or loss of the atrium or bulbus arteriosus (Figure 2B). Hearts dissected maintain their structural integrity and are suitable for histology (Figure 2C</st...

Watch this Scientific Journal Video about Recovery of Adult Zebrafish Hearts for High-throughput Applications at JoVE.com

Recovery of Adult Zebrafish Hearts for High-throughput Applications

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.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and ...

Research

JoVE Journal

Biology

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

A High-Throughput Method For Zebrafish Sperm Cryopreservation and In Vitro Fertilization

This is a method for zebrafish sperm cryopreservation that is an adaptation of the Harvey method (Harvey et al., 1982).  We have introduced two changes to ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

Blood Collection for Biochemical Analysis in Adult Zebrafish

The zebrafish has been used as an animal model for studies of several human diseases. It can serve as a powerful preclinical platform for studies of ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

High-throughput Screening for Chemical Modulators of Post-transcriptionally Regulated Genes

Both transcriptional and post-transcriptional regulation have a profound impact on genes expression. However, commonly adopted cell-based screening assays ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...