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 at takke medlemmerne af Dowling lab (Aaron Reifler, Trent Waugh, Angela Busta, og William Telfer), der bidrog til udviklingen af ​​teknik og produktion af manuskriptet. Dette arbejde blev finansieret af Taubman Institute, Department of Pediatrics ved University of Michigan, og dels fra tilskud fra Muskelsvindfonden Association (JJD MDA186999) og National Institutes of Health (JJD 1K08AR054835).

1.. Fremstilling af poly-L-lysin Coated Dækglas (tid: 1 time) Coating dækglas giver mulighed for hurtig myofiber bosættelse og vedhæftning. Dette kan udføres under dissociation trin af myofiber isolation (trin 2 nedenfor). <ol><li> Klip og placere Parafilm på bunden af ​​en 60 mm petriskål (alle mærker). </li><li> Placer mikroskop dækglas glider (12 mm diameter) på Parafilm i en 60 mm vævskulturskål eller placere dem enkeltvis i enkelt brønde af 24-brønds plader. </li></o...

<ol>
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Genet. 14, 1727-1743 (2005).</li><li>Schredelseker, 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=The+beta+1a+subunit+is+essential+for+the+assembly+of+dihydropyridine-receptor+arrays+in+skeletal+muscle.">The beta 1a subunit is essential for the assembly of dihydropyridine-receptor arrays in skeletal muscle.</a> Proc. Natl. Acad. Sci. U.S.A. 102, 17219-17224 (2005).</li><li>Telfer, W. R., Nelson, D. D., Waugh, T., Brooks, S. V., Dowling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=neb:+a+zebrafish+model+of+nemaline+myopathy+due+to+nebulin+mutation.">neb: a zebrafish model of nemaline myopathy due to nebulin mutation.</a> Dis. Model Mech. 5, 389-396 (2012).</li><li>Dowling, J. J., Low, S. E., Busta, A. S., Feldman, E. 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Zebrafisk er en kraftfuld hvirveldyr model system til at studere muskel udvikling og funktion in vivo 25,27,28. De har også vist sig som et værdifuldt aktiv for modellering af menneskelige muskelsygdomme 14,15,20,29. Selv om der er taget store skridt til at fremme brug og anvendelse af zebrafisk for studiet af muskel funktion og muskel sygdom, der er en konstant kritisk behov for at udvikle værktøjer, der vil give mere tilbundsgående analyse, komplimenter genetiske, morfologiske, adf?...

Skeletmuskel er en højt specialiseret væv er ansvarlig for generering af kontraktile nødvendige styrker til motilitet. Sammentrækning initieres gennem en proces, der kaldes excitation-kontraktion (EF) kobling, der konverterer elektriske signaler til calcium frigivelse fra intracellulære lagre 1,2. Intracellulært calcium release aktiverer sarkomer at afkorte og generere kraft. De mange specifikke komponenter af molekylære maskineri forestår formidlingsarbejdet neuromuskulær transmission 3, EF kobling 4,5, og actin-myosin afhængige sammentrækninger 6 fortsat være den løbende genstand for intens forskning. Endvidere har proteiner, der stabiliserer muskelmembranernes under sammentrækning 7,8 og som medierer signalering mellem myofiber og den ekstracellulære matrix 7,9 blevet identificeret og undersøgt i detaljer. Mutationer i en række gener, der er vigtige for muskel struktur ennd funktion er blevet identificeret som årsag til human skeletmuskulatur sygdomme ( <a href="http://www.musclegenetable.org/" target="_blank">http://www.musclegenetable.org/</a> ). Disse sygdomme, klassificeret bredt som skelet muskellidelser og muskeldystrofier baseret på kliniske og histopatologiske funktioner, der er forbundet med muskelsvaghed, livslang invaliditet og tidlig dødelighed 10,11. Zebrafisken har vist sig at være en fremragende system til modellering og studere human skeletmuskulatur sygdomme 8,12,13. Det er blevet ansat til at validere nye genmutationer 8, definere nye aspekter af sygdommen patofysiologi 14,15, og identificere nye terapeutiske tilgange 15,16. Den effekt af zebrafisk til at studere menneskelig muskelsygdom vedrører det store antal afkom, den hurtige udvikling af muskel struktur og funktion, den optiske klarhed zebrafisk foster, og den lethed af genetiske og farmakologiske manipulation af udviklingslandene zebrafisk 17. Vi og andre 12,18,19 har for nylig udviklet en simpel teknik til hurtig og effektiv isolering af myofibre fra udviklingslandene zebrafisk. Denne metode har muliggjort en undersøgelse af myofibre nærmere, end der kan ydes af hele embryo analyse. Teknikken har været udnyttet til karakterisering af protein subcellulære lokalisering 20, samt til identifikation af vigtige histopatologiske karakteristika som en del af valideringsundersøgelser i nyudviklede sygdomsmodeller 21. Desuden kan isolerede myofibre desuden bruges til direkte billedvisning og elektrofysiologiske studier 22, teknikker, der giver mulighed for afhøring af centrale aspekter af muskelfunktion. Den specifikke protokol for myofiber isolation, sammen med to eksempler på efterfølgende analytiske eksperimenter, er nærmere beskrevet i den resterende del af dette manuskript.

<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>
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</table>

analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations

Zebrafisken har vist sig at være en værdifuld model for at udforske skeletmuskulatur funktion og for at studere humane muskelsygdomme. Trods de mange fordele ved in vivo-analyse af skeletmuskel i zebrafisk, visualisere komplekset og fint struktureret protein miljø ansvarlige for muskelfunktion, især i hele embryoner, kan være problematisk. Denne hindring stammer fra den lille størrelse af zebrafisk skeletmuskulatur (60 um) og den endnu mindre størrelse af sarkomer. Her beskriver vi, og vise en enkel og hurtig metode til at isolere skelet myofibre fra zebrafisk embryoner og larver. Vi har også omfatter protokoller, som illustrerer efterfølgende forberedelse teknikker er nyttige til analyse muskel struktur og funktion. Konkret har vi detalje den efterfølgende immuncytokemiske lokalisering af skeletmuskulaturen proteiner og kvalitativ analyse af stimuleret calcium frigivelse via live celle calcium imaging. Samlet set denne videoartikel giver en ligetil og effektiv fremgangsmåde til isolering og karakterisering af zebrafisk skelet myofibre, en teknik, som tilvejebringer en ledning for myriader efterfølgende undersøgelser af muskel struktur og funktion.

Fluorescerende immunmærkning af myofibre (figur 2) Billeder, der viser forventede fluorescerende mærkning mønster fra myofibre immunofarvede efter vellykket isolering og plettering. De myofibre er blevet mærket med enten anti-ryanodine receptor (1:100) (figur 2A) eller anti-α-actinin (1:100) (figur 2B) antistoffer og viser immunfarvning af triaden og Z-båndet hhv. Sekundære antistoffer blev anvendt, var Alexa Fluor 555 (1:500...

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

Zebrafisk er en voksende system til modellering af humane forstyrrelser i skeletmuskulatur. Vi beskriver en hurtig og effektiv metode til at isolere skeletmuskulaturen myofibre fra embryonale og larve zebrafisk. Denne metode giver en høj densitet myofiber forberedelse egnet til undersøgelse af enkelt skeletmuskulatur fiber morfologi, protein subcellulære lokalisering, og muskel fysiologi.

Analyse af embryonale og larvestadiet Zebrafisk Skeletal myofibre fra Dissocierede 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

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 ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Laser-Påført skade af zebrafisk Embryonale skeletmuskulatur

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

Isolering og dyrkning af individuelle myofibre og deres satellit celler fra voksne skeletmuskler

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

Imaging og 3D Genopbygning cerebrovaskulær Strukturer i embryonale zebrafisk

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