Name: analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations
Uploaded: 2013-11-13T19:30:00
Description: Watch this Scientific Journal Video about Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations at JoVE.com

Forfatterne ønsker å takke medlemmene i Dowling lab (Aaron Reifler, Trent Waugh, Angela Busta, og William Telfer) som bidro til utviklingen av teknikken og til produksjon av manuskriptet. Dette arbeidet ble finansiert av Taubman institutt, Institutt for pediatri ved University of Michigan, og dels fra tilskudd fra muskeldystrofi Association (JJD MDA186999) og National Institutes of Health (JJD 1K08AR054835).

En. Forberedelse av Poly-L-lysin Coated Dekk (Tid: 1 time) Coating Dekk muliggjør rask myofiber ling og heft. Dette kan bli utført i løpet av dissosiasjon trinn av myofiber isolert (trinn 2 nedenfor). <ol><li> Klipp og plassere Parafilm på bunnen av en 60 mm petriskål (alle merker). </li><li> Plasser mikroskop dekkglass slips (12 mm diameter) på Parafilm i en 60 mm vev kultur parabolen eller plassere dem individuelt i enkeltbrønner av 24-brønners plater. </li></ol><p class="jove_co...

<ol>
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I., Zon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+the+Zebrafish+system.">Overview of the Zebrafish system.</a> Methods Cell Biol. 59, 3-10 (1999).</li><li>Nixon, S. J., 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+as+a+model+for+caveolin-associated+muscle+disease;+caveolin-3+is+required+for+myofibril+organization+and+muscle+cell+patterning.">Zebrafish as a model for caveolin-associated muscle disease; caveolin-3 is required for myofibril organization and muscle cell patterning.</a> Hum. Mol. 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Sebrafisk er en kraftig virveldyr modellsystem for å studere muskelutvikling og funksjon in vivo 25,27,28. De har også dukket opp som en verdifull ressurs for å modellere menneskelige muskelsykdommer 14,15,20,29. Mens store fremskritt har blitt tatt for å fremme bruk og anvendelse av sebrafisk for studier av muskelfunksjon og muskelsykdommer, er det et konstant kritisk behov for å utvikle verktøy som vil tillate mer grundig analyse som compliments genetisk, morfologiske, atferdsmessi...

Skjelettmuskel er et høyt spesialisert vev ansvarlig for å generere de kontraktile krefter som er nødvendige for motilitet. Sammentrekning er initiert gjennom en prosess som kalles eksitasjon-kontraksjon (EF) kobling som konverterer elektriske signaler til kalsium utgivelse fra intracellulære butikker 1,2. Intracellulær kalsium utgivelsen aktiverer sarcomere å forkorte og generere kraft. De mange spesifikke komponenter av molekylære maskiner ansvarlig for formidling av nevromuskulær veikryss overføring 3, EC kopling 4,5, og aktin-myosin avhengige sammentrekninger 6 fortsette å være den pågående gjenstand for intens forskning. I tillegg er proteiner som stabiliserer muskelmembranen under sammentrekning 7,8, og som formidler signalering mellom myofiber og den ekstracellulære matriks 7,9 Det er oppdaget og undersøkt i stor detalj. Mutasjoner i en rekke gener som er viktige for muskelstruktur ennd funksjon har blitt identifisert som årsaker til menneskelige skjelettmuskelsykdommer ( <a href="http://www.musclegenetable.org/" target="_blank">http://www.musclegenetable.org/</a> ). Disse sykdommene, klassifisert bredt som skjelett myopatier og muskeldystrofier basert på kliniske og histopatologiske funksjoner, er assosiert med muskelsvakhet, livslang uførhet og tidlig død 10,11. Sebrafisk har vist seg å være en fremragende system for modellering og studere menneskelige skjelettmuskelsykdommer 8,12,13. Det har blitt ansatt for å validere nye genmutasjoner 8, definere nye aspekter av sykdoms patofysiologien 14,15, og identifisere nye terapeutiske tilnærminger 15,16. Kraften av sebrafisk for å studere menneskelig muskel sykdom relatert til det store antall avkom, den raske utviklingen av muskel struktur og funksjon, den optiske klarhet i sebrafisk embryo, og den enkle genetiske og farmakologisk manipulasjon av utviklings sebrafisk 17. Vi og andre 12,18,19 har nylig utviklet en enkel teknikk for rask og effektiv isolering av myofibers fra utviklingssebrafisk. Denne metodikken har aktivert undersøkelsen av myofibers i større detalj enn det som kan tilbys av hele embryo analyse. Teknikken har vært utnyttet for karakterisering av protein subcellulære lokalisering 20, så vel som for identifisering av histopatologiske viktige egenskaper som en del av valideringsstudier i nyutviklede sykdomsmodeller 21. Videre kan isolerte myofibers i tillegg bli brukt for direkte avbildning og for elektrofysiologiske studier 22, teknikker som gir mulighet for avlesning av de viktigste aspektene av muskelfunksjonen. Den spesifikke protokollen for myofiber isolasjon, sammen med to eksempler på senere analytiske eksperimenter, er detaljert i de resterende delene av dette manuskriptet.

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<tr>
			<td>24-well culture plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr>
			<td>10x PBS</td>
			<td>Invitrogen Gibco</td>
			<td>70011</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 Independent medium</td>
			<td>Invitrogen Gibco</td>
			<td>18045</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase Type II</td>
			<td>Worthington Biochemical</td>
			<td>LS004186</td>
			<td>Lot 41H12764</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington Biochemical</td>
			<td>L5004188</td>
			<td></td>
		</tr>
		<tr>
			<td>8% Paraformaldehyde</td>
			<td>Electron Microscopy Sciences</td>
			<td>157-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma</td>
			<td>322415</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma</td>
			<td>X100</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA</td>
			<td>Sigma</td>
			<td>A2153</td>
			<td></td>
		</tr>
		<tr>
			<td>Sheep serum</td>
			<td>Sigma</td>
			<td>S3772</td>
			<td></td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Sigma</td>
			<td>G9023</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass coverslips</td>
			<td>Fischerbrand</td>
			<td>12-545-82 12CIR-1D</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Lysine</td>
			<td>Sigma</td>
			<td>P4707</td>
			<td></td>
		</tr>
		<tr>
			<td>Pronase</td>
			<td>Sigma</td>
			<td>P5147</td>
			<td></td>
		</tr>
		<tr>
			<td>40 &#956;m Filter</td>
			<td>BD Biosciences</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;m Filter</td>
			<td>BD Biosciences</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Prolong Gold antifade reagent</td>
			<td>Invitrogen</td>
			<td>P36931</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-&#945;-Actinin antibody</td>
			<td>Sigma</td>
			<td>A5044</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-RYR antibody</td>
			<td>Abcam</td>
			<td>34C</td>
			<td></td>
		</tr>
		<tr>
			<td>Alexa Fluor antibody</td>
			<td>Invitrogen</td>
			<td>A-21425</td>
			<td></td>
		</tr>
		<tr>
			<td>TWEEN 20</td>
			<td>Sigma</td>
			<td>P1379</td>
			<td></td>
		</tr>
		<tr>
			<td>60 mm Petri dish</td>
			<td>Fischerbrand</td>
			<td>0875713A</td>
			<td></td>
		</tr>
		<tr>
			<td>Poly-L-Ornithine</td>
			<td>Sigma</td>
			<td>P4957</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope slide</td>
			<td>Fischerbrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Caffeine</td>
			<td>Sigma</td>
			<td>C0750</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations

Den sebrafisk har vist seg å være en verdifull modellsystem for å utforske skjelettmuskelfunksjon og for å studere menneskelige muskelsykdommer. Til tross for de mange fordelene som tilbys av in vivo analyse av skjelettmuskulatur i sebrafisk, visualisere komplekse og fint strukturert protein miljø ansvarlig for muskelfunksjonen, særlig i hele embryo, kan være problematisk. Dette hindring stammer fra den lille størrelsen på sebrafisk skjelettmuskulatur (60 mikrometer) og enda mindre størrelse på sarcomere. Her beskriver vi og vise en enkel og rask metode for å isolere skjelett myofibers fra sebrafisk embryo og larver. Vi inkluderer også protokoller som illustrerer legg forberedelse teknikker som er nyttige for å analysere muskel struktur og funksjon. Nærmere bestemt, vi detalj den påfølgende immunocytochemical lokalisering av skjelettmuskelproteiner og kvalitativ analyse av stimulert kalsium utgivelsen via live celle kalsium bildebehandling. Samlet sett denne videoenArtikkelen gir en rett-frem og effektiv metode for isolering og karakterisering av sebrafisk skjelett myofibers, en teknikk som gir en kanal for utallige etterfølgende studier av muskel struktur og funksjon.

Fluorescent immunolabeling av myofibers (figur 2) Bilder som viser forventet fluorescerende merking mønster fra myofibers immunostained etter vellykket isolasjon og platekledning. De myofibers har blitt merket med enten anti-ryanodine reseptor (1:100) (figur 2A), eller anti-α-actinin (1:100) (figur 2B)-antistoffer, og avslører farging av triaden og Z-bånd hhv. Sekundære antistoffer som ble brukt var Alexa Fluor 555 (1:500). Bil...

Watch this Scientific Journal Video about Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations at JoVE.com

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

Sebrafisk er en voksende system for å modellere humane sykdommer i skjelettmuskel. Vi beskriver en rask og effektiv metode for å isolere skjelettmuskel myofibers fra embryo-og larvesebrafisk. Denne metode gir en høy tetthet myofiber preparat egnet for undersøkelse av enkeltskjelettmuskelfiber morfologi, protein subcellulære lokalisering, og muskel-fysiologi.

Analyse av Embryonale og Larvesebrafisk Skeletal myofibers fra Dissosierte Forberedelser

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

Research

JoVE Journal

Biology

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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 ...

Adult and Embryonic Skeletal Muscle Microexplant Culture and Isolation of Skeletal Muscle Stem Cells

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

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

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 ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Farging av dissekerte sebrafisk Embryonale Hjerte

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

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Laser-påført skade av Sebrafisk Embryonic skjelettmuskulatur

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

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Isolasjon og kultur av individuelle myofibers og deres satellitt celler fra voksne Skeletal Muscle

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Bildebehandling og 3D Rekonstruksjon av cerebrovaskulære Structures i Embryonale Sebrafisk

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...