Gli autori desiderano ringraziare i membri del laboratorio Dowling (Aaron Reifler, Trent Waugh, Angela Busta, e William Telfer), che hanno contribuito allo sviluppo della tecnica e alla produzione del manoscritto. Questo lavoro è stato finanziato dall&#39;Istituto Taubman, il Dipartimento di Pediatria presso l&#39;Università del Michigan, e in parte da sovvenzioni dal Distrofia Muscolare (JJD MDA186999) e il National Institutes of Health (JJD 1K08AR054835).

1. Preparazione del Poli-L-lisina Rivestito Coprivetrini (Tempo: 1 ora) Lamelle di rivestimento consente una rapida decantazione myofiber e l&#39;adesione. Ciò può essere eseguita durante la fase di dissociazione dell&#39;isolamento miofibre (passo 2). <ol><li> Tagliare e posizionare Parafilm sul fondo di un piatto 60 millimetri Petri (di qualsiasi marca). </li><li> Posizionare scivola vetro di copertura microscopio (diametro 12 mm) su parafilm in un piatto di coltura tissutale a 60 mm o posizionare singolarmente in singoli pozzetti di piastre da 24 pozzetti. </li></ol> Nota 1: Questi piccoli coprioggetto rotondi aiutano a concentrarsi myofiber numeri e ridurre l&#39;utilizzo di anticorpi in eccesso. Nota 2: Altri vetrini di dimensioni lavoreranno, tuttavia, i volumi di reagenti e di embrioni utilizzati dovranno essere adeguati di conseguenza. <ol start="3"><li> Pipettare 50-200 microlitri di soluzione di poli-L-lisina (0,01%) su ciascun vetrino coprioggetto nella capsula di Petri. </li><li> Lasciare i coprioggetti di sedersi almeno una hour a 37 ° C. Incubazione più lungo non influenzeranno negativamente i risultati. </li><li> Rimuovere poli-L-lisina soluzione dal vetro di copertura e lavare due volte con un minimo di 100 microlitri di PBS 1x. </li><li> Lasciare coprioggetto asciugare. </li><li> Coprivetrini sono ora pronti per miofibre. </li></ol> Nota 3: Al fine di garantire la sterilità, il rivestimento può essere fatto in un cappuccio e su vetrini in autoclave. Nota 4: Mantenere il piatto 60 millimetri Petri con Parafilm sul fondo. Questa configurazione verrà utilizzato durante myofiber placcatura e immunomarcatura (fasi successive). Alternativa 1: invece di lamelle di rivestimento immediatamente prima dell&#39;uso, un vetrino rivestito magazzino può essere utilizzato. Un piatto 60 millimetri Petri contenente un minimo di 2 ml di poli-L-lisina può essere conservato a 4 ° C con numerosi coprioggetto. Con questa alternativa iniziare dal punto 1.5 per elaborare le lamelle per miofibre. Alternativa 2: Abbiamo anche coprioggetto cappotto in poli-L-ornitina. Til suo è più alta intensità di manodopera, ma è utile per la coltura a lungo termine perché vetrini rivestiti di poli-L-ornitina può essere trattato UV. Con il trattamento UV e attenta tecnica sterile myofibers vivi in ​​genere può essere mantenute in coltura 4-7 giorni. 2. Dissociazione di embrioni di zebrafish e placcatura di miofibre (Tempo: da 1 a 3 ore) Nota: il protocollo norma si applica meglio al 3 dpf (giorni dopo la fecondazione) gli embrioni. <ol><li> Trasferire embrioni di zebrafish in un 1,5 ml provetta standard ed eliminare quanta più acqua i pesci in eccesso il più possibile. Tipicamente, possono essere utilizzati 10-20 embrioni per provetta, anche se meno. Utilizzando più di 20-25 embrioni traduce spesso in una preparazione di densità eccessiva. <ol><li> Rimuovere chorions prima di dissociazione, manualmente utilizzando # 5 pinze. In alternativa, la rimozione corion chimica si ottiene con 10-15 minuti di trattamento Pronase. Tipicamente, la rimozione precedente del corion è necessaria solo quando prima conferma di un mutè necessario fenotipo formica o morfologia, o quando si utilizza fasi in cui gli embrioni sono naturalmente tratteggiata da loro corion. </li></ol></li><li> Aggiungere 900 ml di CO 2 media indipendenti per il tubo contenente gli embrioni. </li><li> Aggiungere 100 ml di collagenasi di tipo II, finali concentrazione 3,125 mg / ml (soluzione della collagenasi a 31,25 mg / ml diluito in 1x PBS) per iniziare dissociazione. Collagenasi IV può essere utilizzato anche per la dissociazione. </li><li> Ruotare embrioni in un agitatore orbitale, e triturare ogni 30 min a temperatura ambiente usando una P1000 Pipetman per la triturazione. </li></ol> Nota 2: monitorare attentamente embrione dissociazione, sopra o sotto di dissociazione (soprattutto over) sono le ragioni più comuni per il fallimento del protocollo. I tempi per la digestione variano a seconda dell&#39;intensità di triturazione, il numero di embrioni per provetta, e l&#39;età degli embrioni. E &#39;anche spesso meno (rispetto ai tipi selvatici) per embrioni da mutanti muscolari scheletriche. Appunto3: tempo medio di digestione per età dell&#39;embrione: 1 dpf = 1 ora, 2 dpf = 1,5 ore, 3 dpf = 2 ore e 4 dpf = 2-2,5 ore. <ol start="5"><li> Smettere di digestione quando non interi embrioni sono visibili, ma pezzi solidi sono ancora visibili - questo è essenziale per prevenire overdigestion. </li><li> Provette da centrifuga con embrioni dissociate a 0,8-2,3 g per 3-5 minuti per le cellule pellet. </li><li> Rimuovere il surnatante dalle cellule pellet e lavare 2x con CO 2 media indipendenti. </li><li> Aggiungere CO fresco 2 media indipendenti per risospendere le cellule. 1 ml è tipicamente utilizzata per preparazioni di 10-20 embrioni. Il volume può essere scalata basata sul numero embrione. </li><li> Usando una pipetta P1000, passare sospensione embrione attraverso un filtro da 70 micron. Questo aiuta a rimuovere i residui indesiderati dalla preparazione. </li></ol> Nota 4: sospensione embrione può essere filtrato una seconda volta attraverso un filtro da 40 micron per rimuovere ulteriori residui indesiderati. Da una doppia filtrazione, il recupero è approximdiatamente 800 microlitri da un volume iniziale di 1 ml. <ol start="10"><li> Aggiungere circa 50-100 microlitri di sospensione miofibre ad ogni coprioggetto rivestiti con poli-L-lisina. </li></ol> Nota 5: Eseguire il passaggio 2.9 in piastre di Petri con Parafilm sul fondo, precedentemente preparato. Il Parafilm mantiene la sospensione myofiber di scappare coprioggetto rivestiti. Tenere piastre di Petri coperti per evitare l&#39;evaporazione. <ol start="11"><li> Consentire myofibers di stabilirsi a circa 1 ora sui vetrini rivestiti a temperatura ambiente. </li></ol> Nota 6: myofibers inizieranno a comporre entro 5-10 min. Tuttavia, per buon attacco myofiber, si raccomanda un minimo di 1 ora (1-2 ore). Permettere myofibers di stabilirsi a lungo non danneggia la prep. Per incubazioni più lunghi (notti comprese), antibiotici possono essere aggiunti ai media. Con antibiotici e tecnica sterile fermenti vivi in ​​genere può essere mantenuta per 1-2 giorni. <ol start="12"><li> Live myofibers può osservare a questo punto. Miofibre da embrioni 2 dpf e successivamente sarà visto come cellule striate e allungate (Figura 1). A questo punto, miofibre sono ora pronti per l&#39;analisi diretta o per immunomarcatura. </li></ol> Nota 7: muscolo scheletrico dal 1 dpf embrioni non piastra fibre come allungate e chiaramente striati. Invece, grandi myoballs sono visibili. Inoltre, durante la fase di post dissociazione pellet (punto 2.6), 1 dpf myoballs devono essere centrifugati per un minimo di 8 min a 5000 xg per ottenere un pellet. Per l&#39;analisi degli embrioni in questa fase, si consiglia di utilizzare la linea transgenica che esprime EGFP specificamente nel muscolo scheletrico 23. Ciò consentirà identificazione di cellule di origine muscolare contro altre fonti. 3. Fluorescenza Immunostaining di dissociati Zebrafish miofibre (Durata: circa 1 giorno) 3.1. Immunomarcatura <ol><li> Rimuovere una porzione disupporti da myofibers aderito al vetrino rivestito </li><li> Fissare le cellule con paraformaldeide al 4% o metanolo. Per PFA, rimuovere ½ il volume del supporto e sostituire con la stessa quantità di PFA. Fix per 10 min a RT, quindi rimuovere volume totale, sostituirlo con 50-100 ml di PFA, e fissare per altri 10 min. Per la fissazione alcool, ghiaccio metanolo freddo o acetone può essere applicato a 4 ° C per 10 min o 5 min, rispettivamente. </li><li> Lavare myofibers almeno 3-5x con PBS 1x o 1x PBS più 0,1% di Tween. Il volume medio di lavaggio è 50-100 microlitri per coprioggetto. </li><li> Aggiungere soluzione bloccante a myofibers (Stock di lavoro) e incubare 20-60 minuti a temperatura ambiente. Soluzione bloccante varierà a seconda delle condizioni anticorpi primari e secondari. I due più comuni utilizzate in laboratorio sono (1) 1x PBS, 2 mg / ml BSA, siero di pecora 1%, 0,25% Triton X-100 e finale (2) 0,2% Triton X-100, 2 mg / ml (0,2 % BSA) e siero di pecora / capra 5%. </li><li> Rimuovere soluzione bloccante e aggiungere anticorpo primario diluito in due bsoluzione o PBS bloccaggio. Incubare myofibers notte a 4 ° C. Assicurati di entrambi Parafilm o avvolgere nella pellicola trasparente scatola di Petri contenente i coprioggetti myofiber-caricato rivestite con anticorpi. Piccoli pezzi di carta assorbente inumidito possono essere aggiunti per creare una camera umidificata. </li><li> Rimuovere l&#39;anticorpo primario da coprioggetto e lavare coprioggetto 3-5x per 5 minuti con PBS o la soluzione di blocco. </li><li> Aggiungi anticorpo secondario a concentrazione adeguata diluito in 1x PBS o soluzione di blocco per 1-2 ore a temperatura ambiente. </li><li> Rimuovere anticorpo secondario e lavare myofibers 3-5x in PBS per 5 minuti ciascuno a RT. </li></ol> 3.2. Lamelle di montaggio <ol start="9"><li> Applicare 1-2 gocce di reagente antifade ad un vetrino da microscopio. </li><li> Pick-up con attenzione il vetrino rivestito e immunolabled con pinze e luogo (myofibers giù) il vetrino sul reagente antifade sul vetrino del microscopio. </li></ol> Nota 1: L&#39;attenzione per l&#39;orientamento del cvetrino oated è critica. <ol start="11"><li> Lay 1-2 Kimwipes su una superficie solida dura. Invertire rapidamente la slitta con i vetrini rivestiti riposo sul reagente antifade e posizionarlo (coprioggetto in diminuzione) in Kimwipes. </li><li> Applicare una leggera pressione agli angoli del vetrino da microscopio per rimuovere l&#39;eccesso antifade e formare una perfetta tenuta tra il vetrino e coprioggetto </li><li> Consentire la antifade restante asciugare per 5-10 minuti a temperatura ambiente, poi le miofibre sono pronti ad immagine (Figure 2A e B) o conservare a 4 ° C. </li></ol> 4. Cellulare dal vivo Imaging di calcio Utilizzo GCaMP3 Esperimenti sulle cellule in diretta possono essere eseguite su myofibers prima fissaggio (seguito passo 2.10). Il protocollo che segue descrive l&#39;imaging dal vivo in miofibre esprimono GCaMP3 24, un indicatore di calcio geneticamente codificato, espressa dal muscolo scheletrico specifico zebrafish α-actina promotore (pSKM) 23. Alternativamente,questa tecnica può essere facilmente adattato per usare pigmenti indicatori del calcio, come Fura-2. <ol><li> Iniettare embrioni con pSKM: GCaMP3 costruire nella fase di 1-2 cellule </li><li> A 3 dpf, raccogliere le larve e preparare myofibers come descritto nelle sezioni 1 e 2. Consentire myofibers di aderire per un minimo di 1 ora. Per questa tecnica, vetrini preparano in piatti da 24 pozzetti è necessaria. </li><li> Rimuovere con cura i supporti in eccesso, e aggiungere 300 ml di fresca CO 2 media indipendenti a RT. </li><li> Osservare le cellule sotto un microscopio invertito. GCaMP3 myofibers positivi devono essere visibili sotto fluorescenza verde. </li><li> Installazione della fotocamera per la registrazione, se lo si desidera. </li><li> Preparare una soluzione di caffeina 30 mm di CO 2 media indipendenti. Per stimolare le cellule, pipettare delicatamente 300 ml di soluzione di caffeina nel pozzo sotto ingrandimento. Da 5-10 sec, GCaMP miofibre positivi dovrebbero rispondere agli caffeina con un rapido aumento della fluorescenza (Figura 3). MyofibERS può anche contrarre. Da segnalare, la caffeina induce rilascio di calcio dai depositi del reticolo sarcoplasmatico 25. Altri agenti, come ad esempio KCl 26 e ryanodine 4, possono essere utilizzati per studiare il rilascio di calcio inducibile. </li></ol>

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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=Mutations+affecting+somite+formation+and+patterning+in+the+zebrafish,+Danio+rerio.">Mutations affecting somite formation and patterning in the zebrafish, Danio rerio.</a> Development. 123, 153-164 (1996).</li><li>Gupta, V., 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+zebrafish+dag1+mutant:+a+novel+genetic+model+for+dystroglycanopathies.">The zebrafish dag1 mutant: a novel genetic model for dystroglycanopathies.</a> Hum. Mol. Genet. 20, 1712-1725 (2011).</li><li>Leuranguer, V., Papadopoulos, S., Beam, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+calcium+channel+beta1a+subunits+in+triad+junctions+in+skeletal+muscle.">Organization of calcium channel beta1a subunits in triad junctions in skeletal muscle.</a> J. Biol. Chem. 281, 3521-3527 (2006).</li><li>Capote, J., Bolanos, P., Schuhmeier, R. P., Melzer, W., Caputo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+transients+in+developing+mouse+skeletal+muscle+fibres.">Calcium transients in developing mouse skeletal muscle fibres.</a> J. Physiol. 564, 451-464 (2005).</li><li>Adams, B. A., Tanabe, T., Mikami, A., Numa, S., Beam, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramembrane+charge+movement+restored+in+dysgenic+skeletal+muscle+by+injection+of+dihydropyridine+receptor+cDNAs.">Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs.</a> Nature. 346, (1990).</li><li>Sakowski, S. A., 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=A+novel+approach+to+study+motor+neurons+from+zebrafish+embryos+and+larvae+in+culture.">A novel approach to study motor neurons from zebrafish embryos and larvae in culture.</a> J. Neurosci. Methods. 205, 277-282 (2012).</li><li>Nakano, Y., 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=Biogenesis+of+GPI-anchored+proteins+is+essential+for+surface+expression+of+sodium+channels+in+zebrafish+Rohon-Beard+neurons+to+respond+to+mechanosensory+stimulation.">Biogenesis of GPI-anchored proteins is essential for surface expression of sodium channels in zebrafish Rohon-Beard neurons to respond to mechanosensory stimulation.</a> Development. 137, 1689-1698 (2010).</li><li>Davidson, A. E., 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=Novel+deletion+of+lysine+7+expands+the+clinical,+histopathological+and+genetic+spectrum+of+TPM2-related+myopathies.">Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies.</a> Brain. 136, 508-521 (2013).</li><li>Schredelseker, J., Shrivastav, M., Dayal, A., Grabner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Ca2+-conducting+Ca2++channels+in+fish+skeletal+muscle+excitation-contraction+coupling.">Non-Ca2+-conducting Ca2+ channels in fish skeletal muscle excitation-contraction coupling.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 5658-5663 (2010).</li></ol>

Gli autori dichiarano nessun conflitto di interessi.

Zebrafish sono un potente sistema modello vertebrato per studiare lo sviluppo muscolare e la funzione in vivo 25,27,28. Essi hanno anche emerso come un bene prezioso per la modellazione di malattie muscolari umane 14,15,20,29. Mentre i grandi passi avanti sono stati compiuti per promuovere l&#39;uso e l&#39;applicazione di zebrafish per lo studio della funzione muscolare e delle malattie del muscolo, vi è una necessità critica costante per sviluppare strumenti che consentano più approfo...

Il muscolo scheletrico è un tessuto altamente specializzato responsabile della generazione delle forze necessarie motilità contrattili. La contrazione è iniziata attraverso un processo noto come eccitazione-contrazione (EC) di accoppiamento che converte i segnali elettrici di rilascio di calcio dai depositi intracellulari 1,2. Rilascio di calcio intracellulare attiva il sarcomero di accorciare e generare forza. I numerosi componenti specifici della macchina molecolare responsabile mediare la trasmissione neuromuscolare svincolo 3, accoppiamento EC 4,5, e actina-miosina contrazioni dipendenti 6 continuano ad essere oggetto continuo di intensa ricerca. Inoltre, le proteine ​​che stabilizzano la membrana del muscolo durante la contrazione 7,8 e che mediano la segnalazione tra il myofiber e la matrice extracellulare 7,9 sono stati identificati e studiati nei minimi dettagli. Mutazioni in un certo numero di geni importanti per una struttura muscolarefunzione nd sono stati identificati come causa di malattie del muscolo scheletrici umani ( <a href="http://www.musclegenetable.org/" target="_blank">http://www.musclegenetable.org/</a> ). Queste malattie, classificati in linea di massima come miopatie scheletriche e distrofie muscolari basati su caratteristiche cliniche e istopatologiche, sono associati a debolezza muscolare, invalidità permanente e 10,11 mortalità precoce. Lo zebrafish ha dimostrato di essere un sistema eccezionale per la modellazione e lo studio delle malattie del muscolo scheletrici umani 8,12,13. E &#39;stato impiegato per validare nuove mutazioni del gene 8, definire nuovi aspetti della malattia fisiopatologia 14,15, e identificare nuovi approcci terapeutici 15,16. La potenza della zebrafish per studio delle malattie muscolo umano riferisce al maggior numero di figli, il rapido sviluppo della struttura muscolare e funzione, la chiarezza ottica dell&#39;embrione zebrafish, e la facilità di manipolazione genetica e farmacologica della zebra sviluppopesce 17. Noi e altri 12,18,19 recentemente sviluppato una tecnica semplice per l&#39;isolamento rapido ed efficiente miofibre dal zebrafish sviluppo. Questo metodo ha permesso l&#39;esame di miofibre in maggior dettaglio che può essere fornita da intera analisi embrione. La tecnica è stata sfruttata per la caratterizzazione della proteina localizzazione subcellulare 20 nonché per l&#39;identificazione di importanti caratteristiche istopatologiche come parte di studi di validazione in modelli di malattia di nuova concezione 21. Inoltre, miofibre isolate possono inoltre essere utilizzati per l&#39;imaging dal vivo e per gli studi elettrofisiologici 22, tecniche che consentono l&#39;interrogatorio di aspetti fondamentali della funzione muscolare. Il protocollo specifico per l&#39;isolamento miofibre, insieme a due esempi di esperimenti successivi analitiche, è dettagliata nelle parti rimanenti di questo manoscritto.

<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

Lo zebrafish ha dimostrato di essere un sistema valido modello per esplorare la funzione del muscolo scheletrico e per lo studio delle malattie muscolari umane. Nonostante i numerosi vantaggi offerti da analisi in vivo del muscolo scheletrico nel zebrafish, visualizzando l&#39;ambiente proteina complesso e finemente strutturato responsabile per la funzione muscolare, soprattutto in embrioni interi, può essere problematico. Questo ostacolo deriva dalla piccola dimensione del muscolo scheletrico zebrafish (60 micron) e le dimensioni ancora più piccolo del sarcomero. Qui descriviamo e dimostrare un metodo semplice e rapido per isolare miofibre scheletriche da embrioni di zebrafish e larve. Abbiamo anche i protocolli che illustrano le tecniche di preparazione post utili per analizzare la struttura e la funzione muscolare. In particolare, abbiamo dettaglio la successiva localizzazione immunocitochimica delle proteine ​​dei muscoli scheletrici e l&#39;analisi qualitativa del rilascio del calcio stimolato via live Calcio Imaging cellulare. Nel complesso, questo videoarticolo fornisce un metodo straight-forward ed efficace per l&#39;isolamento e la caratterizzazione di miofibre scheletriche zebrafish, una tecnica che prevede un condotto per la miriade di successivi studi di struttura e funzione muscolare.

Immunomarcatura fluorescente di miofibre (Figura 2) Immagini che mostrano atteso modello di marcatura fluorescente da myofibers immunostained dopo l&#39;isolamento di successo e placcatura. Le miofibre sono stati etichettati sia con anti-recettore ryanodine (1:100) (Figura 2A) o anti-α-actinina (1:100) (Figura 2B) anticorpi, e rivelare rispettivamente immunocolorazione della triade e la Z-band. Anticorpi secondari utilizzati erano ...

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

Zebrafish sono un sistema emergente per modellare disturbi umani del muscolo scheletrico. Descriviamo un metodo veloce ed efficace per isolare miofibre muscolari scheletriche da zebrafish embrionale e larvale. Questo metodo fornisce una preparazione ad alta densità miofibre adatto per lo studio della morfologia singola fibra muscolare scheletrica, proteina localizzazione subcellulare, e fisiologia muscolare.

Analisi delle embrionali e larvali Zebrafish scheletriche miofibre da Preparazioni dissociata

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

analysis-embryonic-larval-zebrafish-skeletal-myofibers-from

Research

JoVE Journal

Biology

11.5K Views.  University of Michigan.  Zebrafish sono un sistema emergente per modellare disturbi umani del muscolo scheletrico. Descriviamo un metodo veloce ed efficace per isolare miofibre muscolari scheletriche da zebrafish embrionale e larvale. Questo metodo fornisce una preparazione ad alta densità miofibre adatto per lo studio della morfologia singola fibra muscolare scheletrica, proteina localizzazione subcellulare, e fisiologia muscolare. 

Video: Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

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 from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

Primaria dissociato mesencefalo colture cellulari dopamina da roditori neonati

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

Ricerca

Biologia

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 to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Isolamento RNA da embrionali Sintesi Zebrafish e cDNA per l&#39;analisi di espressione genica

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 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.

Microdissezione dei tessuti oculari Zebrafish embrionali

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 robust and reliable method for isolating relatively large numbers of proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. The authors have used micro-dissected explant cultures to analyse the growth characteristics of skeletal muscle cells in wild-type and dystrophic muscles. Each of the components of tissue growth, namely cell survival, proliferation, senescence and differentiation can be analysed separately using the methods described here. The net effect of all components of growth can be established by means of measuring explant outgrowth rates. The micro-explant method can be used to establish primary cultures from a wide range of different muscle types and ages and, as described here, has been adapted by the authors to enable the isolation of embryonic skeletal muscle precursors.
Uniquely, micro-explant cultures have been used to derive clonal (single cell origin) skeletal muscle stem cell (SMSc) lines which can be expanded and used for in vivo transplantation. In vivo transplanted SMSc behave as functional, tissue-specific, satellite cells which contribute to skeletal muscle fibre regeneration but which are also retained (in the satellite cell niche) as a small pool of undifferentiated stem cells which can be re-isolated into culture using the micro-explant method.

The micro-dissected explants technique is a robust and reliable method for isolating proliferative skeletal muscle cells from juvenile, adult or embryonic muscles as a source of skeletal muscle stem cells. Uniquely, these cells have been clonally derived to produce skeletal muscle stem cell lines used for in vivo transplantation.

Adulte e embrionali muscolo scheletrico Microexplant Cultura e isolamento di cellule staminali del muscolo scheletrico

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

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.

Dissezione di cuore in larvale, Zebrafish giovanile e degli adulti, Danio rerio</em

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

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.

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.

Immunocolorazione di Dissected Cuore Zebrafish embrionali

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

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.

Laser-lesioni del muscolo scheletrico embrionale Zebrafish

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

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

Isolamento e la coltura di miofibre individuali e delle loro cellule satelliti da adulto muscolo scheletrico

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

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Imaging 3D e ricostruzione di strutture cerebrovascolari in Zebrafish embrionali

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

Analysis of Skeletal Muscle Defects in Larval Zebrafish by Birefringence and Touch-evoke Escape Response Assays

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular dystrophies1-3, congenital myopathies4,5, and disruptions in sarcomeric assembly6,7, due to high genomic and structural conservation with mammals8. Muscular disorganization and locomotive impairment can be quickly assessed in the zebrafish over the first few days post-fertilization. Two assays to help characterize skeletal muscle defects in zebrafish are birefringence (structural) and touch-evoked escape response (behavioral).
Birefringence is a physical property in which light is rotated as it passes through ordered matter, such as the pseudo-crystalline array of muscle sarcomeres9. It is a simple, noninvasive approach to assess muscle integrity in translucent zebrafish larvae early in development. Wild-type zebrafish with highly organized skeletal muscle appear very bright amidst a dark background when visualized between two polarized light filters, whereas muscle mutants have birefringence patterns specific to the primary muscular disorder they model. Zebrafish modeling muscular dystrophies, diseases characterized by myofiber degeneration followed by repeated rounds of regeneration, exhibit degenerative dark patches in skeletal muscle under polarized light. Nondystrophic myopathies are not associated with necrosis or regenerative changes, but result in disorganized myofibers and skeletal muscle weakness. Myopathic zebrafish typically show an overall reduction in birefringence, reflecting the disorganization of sarcomeres.
The touch-evoked escape assay involves observing an embryo&#39;s swimming behavior in response to tactile stimulation10-12. In comparison to wild-type larvae, mutant larvae frequently display a weak escape contraction, followed by slow swimming or other type of impaired motion that fails to propel the larvae more than a short distance12. The advantage of these assays is that disease progression in the same fish type can be monitored in vivo for several days, and that large numbers of fish can be analyzed in a short time relative to higher vertebrates.

The zebrafish is now an established and powerful tool for modeling muscular dystrophies, congenital myopathies, and related neuromuscular diseases. Birefringence and touch-evoked escape behavior are two common noninvasive assays used to determine the degree of muscular disorganization and locomotive impairment of zebrafish embryos during early development.