We thank Beninio Jomok for his help in zebrafish husbandry. This work is funded by NIH HL81753.

1. Preparation of slides and humidified chamber
<ol>
	<li>The humidified chamber can be made from a box such as an emptied tip box. Wrap both the chamber and the cover with aluminium foil to protect the samples from light.</li>
	<li>Inside the chamber, put a pile of wet paper towels to maintain the humidity within the chamber, which will prevent the heart samples from drying. Set a small microplate on the top of paper towel as a rack for the slides.</li>
	<li>Draw either lines or circles on the surface of a microscope slide using the immEdge pen to make wells for heart samples with a dimension about 5x5 mm. Let the slides dry.</li>
	<li>Put the slides in the humidified chamber and add 50ul fresh 4% formaldehyde in each well.</li>
</ol>
2. Dissection of embryonic heart
<ol>
	<li>Anesthetize the fish embryo in E3 egg water 7 containing 0.4% tricaine for about 1 minute until fish stop body movement but still have normal heart beating.</li>
	<li>Put a concave microscope slide on the stage of a dissection microscope. Transfer the embryos to the concaved well.</li>
	<li>Adjust the light combination of the dissecting microscope to clearly reveal the heart. Depending on personal preference, it could be either dark field or DIC-like polarized light condition.</li>
	<li>Clean two insulin syringes with 75% ethanol, then dry.</li>
	<li>Adjust the dissection microscope to higher magnification and focus on the heart. Physically fix an embryo by inserting one needle tip into the yolk-somite boundary close to the head. Insert another needle close to the heart region and pull the heart out quickly. If the heart is not totally separated from the embryo, cut off the remaining connected tissue between the heart and the yolk.</li>
	<li>Adjust the microscope to lower magnification and focus on the separated heart. Use a 10&mu;l pipetteman and set it to 1&mu;l volume. Apply the tip of the pipetteman to either draw or touch the heart sample, so that the isolated heart will be either within or attached to the surface of the tip.</li>
	<li>Dip the pipette tip in 4% formaldehyde solution on prepared slides and depress the pipette piston to release the heart into the solution. The tension of water surface also helps to release the heart from pipette tip to the solution. Put less than 3 hearts in each well.</li>
</ol>
3. Immunostaining
<ol>
	<li>Close the chamber and put it on a shaker with a gentle rocking movement. Fix the heart sample for 20 minutes at room temperature.</li>
	<li>Wipe off the water on the back of the slides and put the slides on the stage of a dissection microscope for buffer exchanging.</li>
	<li>Dip the tip from a 10&mu;l pipetteman into an empty region in each well and keep the tip as far as possible from heart samples. Take cautions to avoid the heart attaching to the tip, since hearts might move with the water flow when the solution is drawn into the tip. Change the tip localization, if needed.</li>
	<li>Dispose the solution onto a tissue and avoid producing any bubbles in the tip since the heart sample is easily lost in the presence of air bubbles. Keeping the heart samples partially dry for a short time can help the hearts stay attached to the slides.</li>
	<li>Add 50&mu;l PBST to cover the heart sample and incubate in the humidified chamber in a slow rocking movement for 5 min. Repeat the rinse one more time. After this step, the heart samples can be stored in the humidified chamber up to 1 week at 4&deg;C.</li>
	<li>Block the heart sample with 10% sheep serum in PBST for 1 hour at room temperature.</li>
	<li>Incubate the heart sample with primary antibodies, such as 1:200 dilution of &beta;-catenin antibody and 1:200 dilution of mef2 specific antibody, in 10% sheep serum/PBST for 1 hour at room temperature.</li>
	<li>Wash the heart sample with PBST three times (5 min. each) at room temperature.</li>
	<li>Incubate the sample with secondary antibodies, such as 1:1000 dilution of Alexa-tagged anti-mouse and/or anti-rabbit antibodies, in PBST for 0.5 hour.</li>
	<li>Wash the heart samples with PBST three times (5 min. each) at room temperature.</li>
</ol>
4. Imaging
<ol>
	<li>Remove PBST and add 10 &mu;l mounting medium to each well. Rock the chamber for several minutes till the solution becomes clear.</li>
	<li>Remove most of the mounting medium and cover all of the wells in one slide with a microscope cover glass (24x50).</li>
	<li>Image fish heart using Zeiss Axioplan 2 microscope equipped with ApoTome (Carl Zeiss).</li>
</ol>
5. Representative Results
An example of an image which reveals membrane and nuclei of all zebrafish cardiomyocytes is shown in Fig. 1. mef2c and &beta;-catenin antibodies were used together to stain heart samples dissected from 52 hpf zebrafish embryo. While mef2c antibody stains the nuclei of cardiomyocytes, &beta;-catenin antibody reveals the border of each cell 8-10. By adjusting the stage of the compound microscope, images of the heart samples can be adjusted to the same orientation, which will allow the observation of outer curvature and inner curvature in the heart 11. The total cardiomyocyte number, cardiomyocyte cell size, and circularity can then be measured.
Another example which reveals the sarcomeric structure of an embryonic heart is shown in Fig. 2. We performed immunostaining using F59, a myosin antibody, in a dissected embryonic heart. The myofibril network in a whole embryonic heart can be clearly revealed at lower magnification, while the striated band of thick filament can be revealed at higher magnification (Fig 2) 6.
<img alt="Figure 1" src="/files/ftp_upload/3510/3510fig1.jpg" /> 
Figure 1. Immunostaining of mef2 (red) and &beta;-catenin (green) to show the nuclei and outlines of cardiomyocytes in a zebrafish ventricle. Scale bar=10&mu;m
<img alt="Figure 2" src="/files/ftp_upload/3510/3510fig2.jpg" /> 
Figure 2. Immnostaining of myosin to show the thick filament in a zebrafish ventricle at either low magnification (A) or high magnification (B).scale bar=10&mu;m

<ol>
	<li>Shin, J. T., Fishman, 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=From+Zebrafish+to+human:+modular+medical+models.">From Zebrafish to human: modular medical models.</a> Annu. Rev. Genomics Hum. Genet. 3, 311-340 (2002).</li><li>Beis, D., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+cell+biology:+following+the+zebrafish+trend.">In vivo cell biology: following the zebrafish trend.</a> Trends. Cell. Biol. 16, 105-112 (2006).</li><li>Schoenebeck, J. J., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cardiac+development:+Advances+in+imaging+add+new+dimensions+to+the+utility+of+zebrafish+genetics.">Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics.</a> Semin. Cell. Dev. Biol. 18, 27-35 (2007).</li><li>Nasevicius, A., Ekker, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effective+targeted+gene+'knockdown'+in+zebrafish.">Effective targeted gene 'knockdown' in zebrafish.</a> Nat. Genet. 26, 216-220 (2000).</li><li>Dahme, T., Katus, H. A., Rottbauer, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fishing+for+the+genetic+basis+of+cardiovascular+disease.">Fishing for the genetic basis of cardiovascular disease.</a> Dis. Model Mech. 2, 18-22 (2009).</li><li>Huang, W., Zhang, R., Xu, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Myofibrillogenesis+in+the+developing+zebrafish+heart:+A+functional+study+of+tnnt2.">Myofibrillogenesis in the developing zebrafish heart: A functional study of tnnt2.</a> Dev. Biol. 331, 237-249 (2009).</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. , (2007).</li><li>Buckingham, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Muscle:+the+regulation+of+myogenesis.">Muscle: the regulation of myogenesis.</a> Curr. Opin. Genet. Dev. 4, 745-751 (1994).</li><li>Wheelock, M. J., Knudsen, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherins+and+associated+proteins.">Cadherins and associated proteins.</a> Vivo. 5, 505-513 (1991).</li><li>Sun, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+hypertrophy+involves+both+myocyte+hypertrophy+and+hyperplasia+in+anemic+zebrafish.">Cardiac hypertrophy involves both myocyte hypertrophy and hyperplasia in anemic zebrafish.</a> PLoS One. 4, e6596-e6596 (2009).</li><li>Auman, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+modulation+of+cardiac+form+through+regionally+confined+cell+shape+changes.">Functional modulation of cardiac form through regionally confined cell shape changes.</a> PLoS Biol. 5, e53-e53 (2007).</li><li>Chen, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+zebrafish+essential+and+regulatory+myosin+light+chains+reduces+cardiac+function+through+distinct+mechanisms.">Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms.</a> Cardiovasc. Res. 79, 97-108 (2008).</li></ol>

No conflicts of interest declared.

Compared to classic whole mount immunostaining methods, our method has the following advantages. First, much stronger fluorescent signals can be consistently obtained due to improved penetration. In the whole mount immunostaining method, the dense skin tissue surrounding the heart significantly reduced the penetration of many antibodies, resulting in high background in the whole body. This problem is especially severe for embryos older than 3-day post-fertilization (dpf). In contrast, the dissected hearts only consist of...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>tricaine</td>
			<td>Research organics</td>
			<td>3007T</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Polysciences</td>
			<td>04018</td>
		</tr>
		<tr>
			<td>Insulin syringe</td>
			<td>Becton Dickinson</td>
			<td>329461</td>
		</tr>
		<tr>
			<td>Triton-X-100</td>
			<td>Sigma</td>
			<td>T8532</td>
		</tr>
		<tr>
			<td>Anti-&beta;-catenin antibody</td>
			<td>Sigma </td>
			<td>C7207</td>
		</tr>
		<tr>
			<td>Anti-Mef2c antibody</td>
			<td>Santa Cruz Biotech</td>
			<td>SC313</td>
		</tr>
		<tr>
			<td>F59</td>
			<td>Zfin</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Anti-&alpha;-actinin antibody</td>
			<td>Sigma</td>
			<td>A7811</td>
		</tr>
		<tr>
			<td>Anti-Ilk antibody</td>
			<td>Cell signaling</td>
			<td>#3862</td>
		</tr>
		<tr>
			<td>Alexa fluor 568 Goat anti rabbit IgG</td>
			<td>Invitrogen</td>
			<td>A11011</td>
		</tr>
		<tr>
			<td>Alexa fluor 488 Goat anti mouse IgG1</td>
			<td>Invitrogen</td>
			<td>A21121</td>
		</tr>
		<tr>
			<td>Mounting medium for fluorescence</td>
			<td>Vector</td>
			<td>H-1200</td>
		</tr>
		<tr>
			<td>ImmEdge pen</td>
			<td>Vector </td>
			<td>H-4000</td>
		</tr>
		<tr>
			<td>Poly-L-lysin coated slides</td>
			<td>Electron microscopy sciences </td>
			<td>63410-01</td>
		</tr>
		<tr>
			<td>Microscope cover glass</td>
			<td>Fisher </td>
			<td>12-543-D</td>
		</tr>
		<tr>
			<td>Concaved microscope slides</td>
			<td>Fisher </td>
			<td>7-1305-8</td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Leica MZ95</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Compound microscope</td>
			<td>Zeiss</td>
			<td>Axioplan2</td>
		</tr>
		<tr>
			<td>ApoTome</td>
			<td>Zeiss</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>
PBST: 
1 x PBS 
0.5% Triton-X 100

25X Tricaine: 
400 mg Tricaine 
97.9 mL ddH2O 
2.1 mL (1M Tris pH9) 
Adjust pH to 7.0

immunostaining of dissected zebrafish embryonic heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

Watch this Scientific Journal Video about Immunostaining of Dissected Zebrafish Embryonic Heart at JoVE.com

Immunostaining of Dissected Zebrafish Embryonic Heart

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

immunostaining-of-dissected-zebrafish-embryonic-heart

Research

JoVE Journal

Biology

17.6K 조회수.  Mayo Clinic College of Medicine. zebrafish 배아 심장의 immunostaining를 수행하는 빠른 방법이 설명되어 있습니다. 전체 마운트 immunostaining 접근 방식에 비해,이 방법은 크게 크게 감소 처리 시간 이내에 심장에 세포 / subcellular 구조를 공개 고해상도 이미지를 얻는 수있는 항체의 침투를 증가시킵니다.

비디오: Immunostaining of Dissected Zebrafish Embryonic Heart

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

The morphogenesis of the Drosophila embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell shape changes during embryonic development. One of the challenges faced in studying this structure is that the lumen of the heart tube, as well as the membrane features that are crucial to heart tube formation, are difficult to visualize in whole mount embryos, due to the small size of the heart tube and intra-lumenal space relative to the embryo. The use of transmission electron microscopy allows for higher magnification of these structures and gives the advantage of examining the embryos in cross section, which easily reveals the size and shape of the lumen. In this video, we detail the process for reliable fixation, embedding, and sectioning of late stage Drosophila embryos in order to visualize the heart tube lumen as well as important cellular structures including cell-cell junctions and the basement membrane.

Preparation of embryos for Electron Microscopy of the Drosophila embryonic heart tube

We describe a process for fixation, embedding, sectioning, and imaging of late stage Drosophila embryos for Trasmission Electron Microscopy of the embryonic heart tube. This technique allows for the visualization of the heart tube lumen as well as the basement membrane, which lines the lumen of the heart.

The morphogenesis of the Drosophila  embryonic heart tube has emerged as a valuable model system for studying cell migration, cell-cell adhesion and cell ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Whole-mount Immunohistochemical Analysis for Embryonic Limb Skin Vasculature: a Model System to Study Vascular Branching Morphogenesis in Embryo

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching morphogenesis. We have developed the limb skin vasculature model to study vascular development in which a pre-existing primitive capillary plexus is reorganized into a hierarchically branched vascular network. Whole-mount confocal microscopy with multiple labelling allows for robust imaging of intact blood vessels as well as their cellular components including endothelial cells, pericytes and smooth muscle cells, using specific fluorescent markers. Advances in this limb skin vasculature model with genetic studies have improved understanding molecular mechanisms of vascular development and patterning. The limb skin vasculature model has been used to study how peripheral nerves provide a spatial template for the differentiation and patterning of arteries. This video article describes a simple and robust protocol to stain intact blood vessels with vascular specific antibodies and fluorescent secondary antibodies, which is applicable for vascularized embryonic organs where we are able to follow the process of vascular development.

We introduce a whole-mount immunohistochemistry and laser scanning confocal microscopy with multiple labelling for analyzing intricate vascular network formation in mouse embryonic limb skin.

Whole-mount immunohistochemical analysis for imaging the entire vasculature is pivotal for understanding the cellular mechanisms of branching ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...