Name: immunostaining of dissected zebrafish embryonic heart
Uploaded: 2012-01-10T12:00:00
Description: Watch this Scientific Journal Video about Immunostaining of Dissected Zebrafish Embryonic Heart at JoVE.com

Vi takker Beninio Jomok for hans hjælp i zebrafisk dyrehold. Dette arbejde er finansieret af NIH HL81753.

1. Udarbejdelse af dias og befugtes kammer <ol><li> Den befugtet kammer kan foretages fra en kasse, såsom en tømt tip boks. Wrap både kammer og dækslet med aluminiumsfolie for at beskytte prøver fra lys. </li><li> Inde i kammeret, sætte en bunke våde papirservietter til at opretholde fugtigheden i kammeret, hvilket vil forhindre hjertet prøver fra tørring. Indstil en lille mikroplade på toppen af ​​køkkenrulle som et rack til dias. </li><li> Tegn enten linjer eller cirkler på overfladen af ​​et obj...

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	<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>

Sammenlignet med klassiske hele montere immunfarvning metoder, har vores metode følgende fordele. For det første kan meget stærkere fluorescerende signaler være konsistent som følge af forbedret penetration. I hele mount immunfarvning metode, hvilket er betydeligt den tætte hud væv omkring hjertet reduceret udbredelsen af ​​mange antistoffer, hvilket resulterer i en høj baggrund i hele kroppen. Dette problem er især alvorlig for embryoner ældre end 3-dages post-befrugtning (DPF). I modsætning hertil kun d...

<table border="1"><tbody><tr><td> Navn på reagenset </td><td> Firma </td><td> Katalog nummer </td></tr><tr><td> tricaine </td><td> Forskning økologi </td><td> 3007T </td></tr><tr><td> Formaldehyd </td><td> Polysciences </td><td> 04018 </td></tr><tr><td> Insulinsprøjte </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-β-catenin antistof </td><td> Sigma </td><td> C7207 </td></tr><tr><td> Anti-Mef2c antistof </td><td> Santa Cruz Biotech </td><td> SC313 </td></tr><tr><td> F59 </td><td> Zfin </td><td></td></tr><tr><td> Anti-α-actinin antistof </td><td> Sigma </td><td> A7811 </td></tr><tr><td> Anti-ilk antistof </td><td> Celle signalering </td><td> # 3862 </td></tr><tr><td> Alexa fluor 568 gede anti kanin IgG </<html> td&gt; <td> Invitrogen </td><td> A11011 </td></tr><tr><td> Alexa fluor 488 gede anti muse-IgG1 </td><td> Invitrogen </td><td> A21121 </td></tr><tr><td> Montering medium for fluorescens </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 belagt slides </td><td> Elektronmikroskopi videnskaber </td><td> 63410-01 </td></tr><tr><td> Microscope dække glas </td><td> Fisher </td><td> 12 til 543-D </td></tr><tr><td> Konkav mikroskopobjektglas </td><td> Fisher </td><td> 7-1305-8 </td></tr><tr><td> Dissektion mikroskop </td><td> Leica MZ95 </td><td></td></tr><tr><td> Sammensat mikroskop </td><td> Zeiss </td><td> Axioplan2 </td></tr><tr><td> ApoTome </td><td> Zeiss </td><td></td></tr></tbody></table> PBST: 1 x PBS 0,5% Triton-X 100 ENT &quot;&gt; 25X Tricaine: 400 mg Tricaine 97,9 mL Hedeselskabet 2 O 2,1 ml (1M Tris pH9) Justér pH til 7,0

immunostaining of dissected zebrafish embryonic heart

Zebrafisk foster bliver et populært in vivo hvirveldyr model for at studere hjertets udvikling og menneskelig hjertesygdomme på grund af sin fordelagtige embryologi og genetik 1,2. Omkring 100-200 embryoner, som er let tilgængelige hver uge fra et enkelt par af voksne fisk. Den gennemsigtige embryoner, der udvikler ex utero gør dem ideelle til at vurdere hjerte defekter 3. Udtrykket af genet kan manipuleres via morpholino teknologi eller RNA injektion 4. Desuden frem genetiske skærme har allerede genereret en liste over mutanter, der påvirker forskellige perspektiver af cardiogenesis 5. Hele mount immunfarvning er en vigtig teknik i denne dyremodel til at afsløre udtryk mønster af de målrettede protein til et bestemt væv 6. Imidlertid har billeder i høj opløsning, der kan afsløre cellulære eller subcellulære strukturer været vanskeligt, hovedsagelig på grund af fysiske Statusmeddelelseion af hjertet og de fattige penetration af antistoffer. Her præsenterer vi en metode til at imødegå disse flaskehalse ved at dissekere hjerte først og derefter gennemføre farvningen processen på overfladen af ​​et objektglas. For at forhindre tab af lille hjerte prøver og for at lette løsning håndtering, vi begrænset hjertet prøverne inden for en cirkel på overfladen af ​​de objektglas trukket af en immEdge pen. Efter farvning, kan fluorescens signaler direkte iagttages af en sammensat mikroskop. Vores nye metode forbedrer indtrængningen for antistoffer, da et hjerte fra en embryonale fisk kun består af nogle få cellelag. Billeder i høj kvalitet fra intakte hjerter kan opnås inden for en meget reduceret procession tid til zebrafisk embryoner i alderen fra dag 2 til 6 dage. Vores metode kan potentielt udvides til pletten andre organer dissekeret fra enten zebrafisk eller andre små dyr.

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

Immunostaining of Dissected Zebrafish Embryonic Heart

En hurtig måde at gennemføre immunfarvning af zebrafisk embryonale hjerte er beskrevet. Sammenlignet med hele montere immunfarvning tilgang, dramatisk denne metode øger penetration af de antistoffer, der gør det muligt at få billeder i høj opløsning, der afslører cellulære / subcellulære strukturer i hjertet inden for en meget reduceret behandlingstid.

Immunfarvning af dissekerede zebrafisk Embryonale Heart

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

Research

JoVE Journal

Biology

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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 ...

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

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

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

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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

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Et system til<em&gt; Ex vivo</em&gt; Dyrkning af embryonale Pancreas

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

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A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

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