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 ...

<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>

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

17.5K visualizações.  Mayo Clinic College of Medicine. Uma maneira rápida de realizar imunocoloração do coração embrionário zebrafish é descrito. Em comparação com toda a abordagem montagem imunocoloração, este método aumenta drasticamente a penetração dos anticorpos, o que permite obter imagens de alta resolução que revelam estruturas celulares / subcelulares no coração dentro de um tempo de processamento muito reduzido.