Les auteurs tiennent à remercier les membres du laboratoire Dowling (Aaron Reifler, Trent Waugh, Angela Busta, et William Telfer) qui ont contribué au développement de la technique et de la production du manuscrit. Ce travail a été financé par l&#39;Institut Taubman, le département de pédiatrie de l&#39;Université du Michigan, et en partie par des dons de la Muscular Dystrophy Association (JJD MDA186999) et les National Institutes of Health (JJD 1K08AR054835).

Une. lamelles de préparation de poly-L-lysine couché (Durée: 1 h) lamelles de revêtement permet le tassement des fibres musculaires rapides et l&#39;adhérence. Cela peut être effectué au cours de l&#39;étape de dissociation de l&#39;isolement de fibres musculaires (étape 2 ci-dessous). <ol><li> Couper et placer Parafilm sur le fond d&#39;un plat de 60 mm de Pétri (toute marque). </li><li> Placer des bordereaux de verre de couverture de microscope (diamètre 12 mm) sur Parafilm dans une boîte de culture tissulaire de 60 mm ou de les placer individuellement dans des puits individuels de plaques à 24 puits. </li></ol> Note 1: Ces petites lamelles rondes aident à concentrer le nombre des fibres musculaires et réduire l&#39;utilisation de l&#39;excès d&#39;anticorps. Note 2: D&#39;autres lamelles de taille vont travailler, mais les volumes de réactifs et d&#39;embryons utilisés devront être ajustés en conséquence. <ol start="3"><li> Introduire à la pipette 50 à 200 ul de solution de poly-L-lysine (0,01%) sur chaque lamelle couvre-objet dans la boîte de Pétri. </li><li> Autoriser les lamelles de s&#39;asseoir au moins un hour à 37 ° C. Incubations plus longues ne seront pas influencer négativement les résultats. </li><li> Retirer poly-L-lysine solution du couvercle en verre et laver deux fois avec un minimum de 100 pi 1x PBS. </li><li> Permettre aux lamelles de sécher. </li><li> Lamelles sont maintenant prêts pour les fibres musculaires. </li></ol> Note 3: Afin de garantir la stérilité, le revêtement peut être fait dans une hotte et sur des lamelles en autoclave. Note 4: Gardez la boîte de Pétri de 60 mm avec du Parafilm sur le fond. Cette configuration sera utilisée pendant myofiber placage et immunomarquage (plus tard étapes). Variante 1: Au lieu de lamelles de revêtement immédiatement avant de les utiliser, une lamelle de papier couché peut être utilisé. Un plat de 60 mm de Pétri contenant un minimum de 2 ml de poly-L-lysine peut être conservé à 4 ° C contenant de nombreuses lamelles. Avec cette alternative commencer à l&#39;étape 1.5 pour traiter les lamelles de fibres musculaires. Alternative 2: Nous avons également des lamelles de manteau en poly-L-ornithine. Tson est plus de travail, mais est utile pour plus de culture terme parce lamelles revêtues de poly-L-ornithine peut être traité anti-UV. Avec un traitement UV et technique stérile attention myofibers vivants peuvent généralement être maintenues en culture provenant 4-7 jours. 2. La dissociation des embryons de poisson zèbre et de placage de fibres musculaires (Durée: 1 à 3 heures) Remarque: le protocole standard s&#39;applique le mieux à 3 dpf (jours après la fécondation) des embryons. <ol><li> Transfert d&#39;embryons de poisson zèbre dans un tube de 1,5 ml centrifugeuse standard et éliminer autant d&#39;eau de poissons excès que possible. Typiquement, 10 à 20 embryons par tube, bien que moins peuvent être utilisés. Utilisation de plus de 20 à 25 embryons se traduit souvent par une préparation de densité excessive. <ol><li> Retirer chorions avant dissociation, manuellement à l&#39;aide # 5 forceps. En variante, l&#39;élimination du chorion chimique est réalisée avec un traitement Pronase 10-15 min. En règle générale, la suppression de la précédente chorion n&#39;est nécessaire que si la confirmation préalable d&#39;un mutphénotype fourmi ou la morphologie est nécessaire, ou lorsque vous utilisez les stades où les embryons sont naturellement éclosion de leur chorion. </li></ol></li><li> Ajouter 900 pi de CO 2 médias indépendants dans le tube contenant les embryons. </li><li> Ajouter 100 pi de type II de la collagénase, concentration finale de 3,125 mg / ml (solution mère de la collagénase à 31,25 mg / ml dilué dans du PBS 1X) pour commencer la dissociation. Collagénase IV peut également être utilisé pour la dissociation. </li><li> Faire tourner les embryons sur un agitateur orbital, et triturer toutes les 30 min à température ambiante en utilisant un Pipetman P1000 pour la trituration. </li></ol> Note 2: Surveiller attentivement embryon dissociation; plus ou moins de dissociation (surtout plus) sont les raisons les plus fréquentes de l&#39;échec du protocole. Temps pour la digestion varie en fonction de l&#39;intensité de trituration, nombre d&#39;embryons par tube, et l&#39;âge de l&#39;embryon. Il est également souvent moins (par rapport au type sauvage) à partir d&#39;embryons mutants du muscle squelettique. Remarque3: temps de digestion moyen par âge de l&#39;embryon: 1 dpf = 1 h, 2 dpf = 1,5 h, 3 dpf = 2 h et 4 dpf = 2-2,5 h. <ol start="5"><li> Arrêtez la digestion quand aucun des embryons entiers sont visibles, mais des morceaux solides sont encore visibles - ce qui est essentiel pour éviter une digestion excessive. </li><li> Tubes à centrifuger avec des embryons dissociées à 0,8 à 2,3 g pendant 3-5 min à culot de cellules. </li><li> Retirer le surnageant de culot de cellules et laver 2x CO 2 médias indépendants. </li><li> Ajouter CO frais 2 médias indépendants à remettre les cellules. 1 ml est généralement utilisé pour préparations de 10-20 embryons. Le volume peut être adapté en fonction du nombre d&#39;embryons. </li><li> Avec une pipette P1000, passer embryon suspension à travers un filtre de 70 um. Cela permet de supprimer les débris indésirables de la préparation. </li></ol> Note 4: Embryon suspension peut être filtrée une seconde fois à travers un filtre de 40 um pour éliminer davantage les débris indésirables. D&#39;une double filtration, la récupération est approximdiatement 800 ul d&#39;un volume initial de 1 ml. <ol start="10"><li> Ajouter environ 50 à 100 ul de suspension de fibres musculaires à chaque lamelle couvre-objet revêtues de poly-L-lysine. </li></ol> Note 5: Effectuer l&#39;étape 2.9 dans les boîtes de Pétri avec du Parafilm sur le fond, préalablement préparé. Le Parafilm maintient la suspension de fibres musculaires de courir au large des lamelles recouvertes. Gardez boîtes de Pétri couverts pour éviter l&#39;évaporation. <ol start="11"><li> Permettent de régler les fibres musculaires d&#39;environ 1 heure sur les lamelles recouvertes à la température ambiante. </li></ol> Note 6: les fibres musculaires commencent à s&#39;installer dans 5-10 min. Cependant, pour une bonne fixation de fibres musculaires, un minimum de 1 heure (1-2 heures) est recommandé. Permettre fibres musculaires à se contenter de plus ne nuira pas à la préparation. Pour les incubations plus longues (y compris la nuit), les antibiotiques peuvent être ajoutés aux médias. Avec des antibiotiques et technique stérile cultures vivantes peuvent généralement être conservés pendant 1-2 jours. <ol start="12"><li> Direct myofibers peut être observé à ce stade. Fibres musculaires à partir d&#39;embryons 2 dpf et plus tard sera considéré comme cellules striées et allongées (figure 1). À ce stade, les fibres musculaires sont maintenant prêts pour l&#39;analyse en temps réel ou pour immunomarquage. </li></ol> Note 7: Le muscle squelettique de 1 dpf embryons ne pas plaquer les fibres allongées et clairement que striés. Au lieu de cela, les grandes myoballs sont visibles. En outre, au cours de l&#39;après dissociation de phase de granulation (étape 2.6), 1 dpf myoballs doivent être centrifugé pendant un minimum de 8 min à 5000 xg pour obtenir une pastille. Pour l&#39;analyse des embryons à ce stade, il est recommandé d&#39;utiliser la lignée transgénique exprimant EGFP spécifiquement dans le muscle squelettique 23. Ceci permettra l&#39;identification des cellules d&#39;origine musculaire par rapport à d&#39;autres sources. 3. Fluorescence Immunocoloration dissociées de poisson-zèbre fibres musculaires (Durée: environ 1 jour) 3.1. Immunomarquage <ol><li> Retirez une partie demédias de fibres musculaires adhéré à la lamelle enduit </li><li> Fixer les cellules en utilisant 4% de paraformaldéhyde ou le méthanol. Pour PFA, retirez ½ le volume des médias et la remplacer par la même quantité de PFA. Fixer pendant 10 min à température ambiante, puis retirez volume total, le remplacer par 50-100 ul de PFA, et fixer pour 10 min de plus. Pour la fixation de l&#39;alcool, de la glace ou de l&#39;acétone methanol froid peut être appliqué à 4 ° C pendant 10 min ou 5 min, respectivement. </li><li> Laver les fibres musculaires au moins 3-5x avec PBS 1x ou 1x PBS plus 0,1% de Tween. Volume de lavage moyenne est de 50-100 ul par lamelle. </li><li> Ajouter une solution de blocage de fibres musculaires (langue de travail) et incuber 20-60 min à température ambiante. La solution de blocage varie en fonction de l&#39;anticorps primaire et secondaire. Les deux plus couramment utilisé dans le laboratoire sont (1) 1x PBS, 2 mg / ml de BSA, sérum de mouton de 1%, 0,25% de Triton X-100 final et (2) 0,2% de Triton X-100, 2 mg / ml (0,2 % de BSA) et 5% de sérum ovins / caprins. </li><li> Retirer la solution de blocage et ajouter anticorps primaire dilué dans les deux bsolution PBS ou de verrouillage. Incuber myofibres pendant une nuit à 4 ° C. Assurez-vous que soit Parafilm ou l&#39;envelopper dans du Saran la boîte de Pétri contenant les lamelles de fibres musculaires chargé recouvertes d&#39;anticorps. De petits morceaux de serviettes en papier humidifiées peuvent être ajoutées pour créer une chambre humidifiée. </li><li> Eliminer l&#39;anticorps primaire de lamelles et laver lamelles 3-5x pendant 5 min avec du PBS ou solution de blocage. </li><li> Ajouter anticorps secondaire à une concentration appropriée dilué dans PBS 1x ou solution de blocage pendant 1-2 heures à la température ambiante. </li><li> Eliminer l&#39;anticorps secondaire et laver les fibres musculaires 3-5x dans du PBS pendant 5 min chaque à température ambiante. </li></ol> 3.2. Des lamelles de montage <ol start="9"><li> Appliquer 1-2 gouttes de réactif de antifade sur une lame de microscope. </li><li> Pick-up soigneusement la lamelle enduit et immunolabled aide d&#39;une pince et le lieu (fibres musculaires vers le bas) de la lamelle sur le réactif de antifade sur la lame de microscope. </li></ol> Remarque 1: Attention à l&#39;orientation de la clamelle oated est critique. <ol start="11"><li> Posez 1-2 Kimwipes sur une surface solide dur. Inverser rapidement la lame avec les lamelles recouvertes reposant sur le réactif de antifade et placez-le (lamelle vers le bas) sur la Kimwipes. </li><li> Appliquer une légère pression sur les coins de la lame de microscope pour éliminer l&#39;excès antifade et former un joint étanche entre la lame et lamelle </li><li> Permettre la antifade restant à sécher pendant 5-10 min à température ambiante, puis les fibres musculaires sont prêts à l&#39;image (Figures 2A et B) ou conserver à 4 ° C. </li></ol> 4. Imagerie de cellules vivantes de calcium Utilisation GCaMP3 Expériences sur des cellules vivantes peuvent être effectuées sur les fibres musculaires avant la fixation (ci-après l&#39;étape 2.10). Le protocole suivant décrit l&#39;imagerie en direct dans les fibres musculaires exprimant GCaMP3 24, un indicateur de calcium codé génétiquement, exprimée par le α-actine poisson zèbre promoteur spécifique du muscle squelettique (PSKm) 23. Autrement,cette technique peut facilement être adapté pour utiliser des colorants indicateurs de calcium tels que Fura-2. <ol><li> Injecter embryons de PSKm: GCaMP3 construire au stade 1-2 cellules </li><li> A 3 dpf, recueillir les larves et préparer les fibres musculaires comme décrit dans les sections 1 et 2. Permettre aux fibres musculaires d&#39;adhérer pour un minimum de 1 heure. Pour cette technique, la préparation des lamelles dans des boîtes de 24 puits sont nécessaires. </li><li> Retirez délicatement le support en trop, et ajouter 300 ul de frais CO 2 médias indépendants à la température ambiante. </li><li> Respecter les cellules sous un microscope inversé. Myofibres positives GCaMP3 doivent être visibles sous fluorescence verte. </li><li> Mettre en place la caméra pour l&#39;enregistrement, si vous le souhaitez. </li><li> Préparer une solution de caféine 30 mM de CO 2 médias indépendants. Pour stimuler les cellules, la pipette doucement 300 pi de solution de caféine dans le puits à la loupe. Dans 5-10 sec, les fibres musculaires positives GCaMP doivent répondre à la caféine à une augmentation rapide de la fluorescence (figure 3). Myofibteurs peuvent aussi contracter. Fait à noter, la caféine induit la libération de calcium dans les magasins du réticulum sarcoplasmique 25. D&#39;autres agents, tels que KCl 26 et ryanodine 4, peuvent être utilisés pour étudier la libération de calcium inductible. </li></ol>

<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>

Les auteurs déclarent aucun conflits d&#39;intérêts.

Le poisson zèbre est un système de modèle vertébré puissant pour étudier le développement musculaire et la fonction in vivo en 25,27,28. Ils ont également vu comme un atout précieux pour la modélisation de maladies musculaires humaines 14,15,20,29. Si de grands progrès ont été prises pour promouvoir l&#39;utilisation et l&#39;application de poisson zèbre pour l&#39;étude de la fonction musculaire et la maladie du muscle, il ya un besoin critique constante de développer des ...

Le muscle squelettique est un tissu hautement spécialisé chargé de générer les forces nécessaires à la motilité contractile. Contraction est initiée par un processus connu sous le nom excitation-contraction (CE) de couplage qui convertit les signaux électriques à la libération de calcium des réserves intracellulaires 1,2. La libération de calcium intracellulaire active le sarcomère à raccourcir et à générer de la force. Les nombreux éléments spécifiques de la machinerie moléculaire responsable de la médiation transmission de la jonction neuromusculaire 3, le couplage CE 4,5, et l&#39;actine-myosine contractions dépendantes 6 continuent à être l&#39;objet permanent de recherche intense. En outre, les protéines qui stabilisent la membrane du muscle lors de la contraction de 7,8 et que la signalisation entre le myofiber et la matrice extracellulaire 7,9 médiate ont été identifiés et étudiés dans les moindres détails. Des mutations dans un certain nombre de gènes importants pour la structure d&#39;un musclefonction e ont été identifiés comme causes de maladies musculaires squelettiques humaines ( <a href="http://www.musclegenetable.org/" target="_blank">http://www.musclegenetable.org/</a> ). Ces maladies, classés largement que myopathies squelettiques et les dystrophies musculaires basés sur les caractéristiques cliniques et histopathologiques, sont associées à une faiblesse musculaire, une incapacité permanente, et au début de 10,11 de mortalité. Le poisson zèbre s&#39;est avéré être un excellent système pour la modélisation et l&#39;étude des maladies des muscles squelettiques humains 8,12,13. Il a été utilisé pour valider de nouvelles mutations génétiques 8, définir de nouveaux aspects de la maladie physiopathologie 14,15, et d&#39;identifier de nouvelles approches thérapeutiques 15,16. La puissance de poisson zèbre pour étudier la maladie du muscle humain concerne le grand nombre d&#39;enfants, le développement rapide de la structure musculaire et de la fonction, la clarté optique de l&#39;embryon de poisson zèbre, et la facilité de la manipulation génétique et pharmacologique du zèbre en développementpoissons 17. Nous et les autres 12,18,19 avons récemment développé une technique simple pour l&#39;isolement rapide et efficace des fibres musculaires du poisson zèbre en développement. Cette méthodologie a permis l&#39;examen des fibres musculaires plus en détail que peut être fournie par l&#39;analyse de l&#39;embryon entier. La technique a été exploitée pour la caractérisation de la protéine 20 localisation sous-cellulaire ainsi que pour l&#39;identification des caractéristiques histopathologiques importants dans le cadre d&#39;études de validation dans des modèles de maladie nouvellement mis au point 21. En outre, les fibres musculaires isolées peuvent en outre être utilisés pour l&#39;imagerie en direct et pour les études électrophysiologiques 22, les techniques qui permettent d&#39;interroger les principaux aspects de la fonction musculaire. Le protocole spécifique pour l&#39;isolation des fibres musculaires, ainsi que deux exemples d&#39;expériences analytiques ultérieurs, est détaillé dans les parties restantes de ce manuscrit.

<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

Le poisson zèbre s&#39;est révélé être un système de modèle utile pour explorer la fonction des muscles squelettiques et pour l&#39;étude de maladies musculaires humaines. Malgré les nombreux avantages offerts par l&#39;analyse in vivo du muscle squelettique chez le poisson zèbre, visualiser le milieu complexe de protéines et finement structuré responsable de la fonction musculaire, en particulier dans les embryons entiers, peut être problématique. Cet obstacle tient à la petite taille du muscle squelettique poisson zèbre (60 um) et la taille encore plus petite du sarcomère. Ici, nous décrivons et démontrons une méthode simple et rapide pour isoler les fibres musculaires squelettiques à partir d&#39;embryons de poisson zèbre et de larves. Nous incluons également des protocoles qui illustrent les techniques de préparation soumettre utiles pour analyser la structure et la fonction musculaire. Plus précisément, nous détaillons la localisation immunocytochimique ultérieure des protéines du muscle squelettique et l&#39;analyse qualitative de la libération de calcium stimulée par l&#39;imagerie en direct de calcium de la cellule. Dans l&#39;ensemble, cette vidéoarticle fournit une méthode straight-forward et efficace pour l&#39;isolement et la caractérisation des fibres musculaires squelettiques de poisson zèbre, une technique qui permet une conduite pour des études ultérieures myriade de structure et la fonction musculaire.

Immunomarquage fluorescent de fibres musculaires (figure 2) Images montrant motif de marquage fluorescent attendu de fibres musculaires immunocolorées après isolement succès et le placage. Les fibres musculaires ont été marquées avec soit anti-récepteur de la ryanodine (1:100) (figure 2A) ou l&#39;anti-α-actinine (1:100) (Figure 2B), les anticorps, et révéler l&#39;immunomarquage de la triade et le Z-bande, respectivement....

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

Poisson zèbre sont d&#39;un nouveau système pour la modélisation de maladies humaines du muscle squelettique. Nous décrivons une méthode rapide et efficace pour isoler les fibres musculaires des muscles squelettiques de poisson zèbre embryonnaire et larvaire. Cette méthode produit une préparation de haute densité myofiber approprié pour l&#39;étude de la morphologie unique de fibres de muscle squelettique, la localisation subcellulaire des protéines, et la physiologie des muscles.

Analyse des embryonnaire et larvaire de poisson-zèbre squelettiques fibres musculaires de préparations dissociées

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

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Biology

11.5K vues.  University of Michigan.  Poisson zèbre sont d'un nouveau système pour la modélisation de maladies humaines du muscle squelettique. Nous décrivons une méthode rapide et efficace pour isoler les fibres musculaires des muscles squelettiques de poisson zèbre embryonnaire et larvaire. Cette méthode produit une préparation de haute densité myofiber approprié pour l'étude de la morphologie unique de fibres de muscle squelettique, la localisation subcellulaire des protéines, et la ph...

Vidéo: 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.

Primaire Dissocié cultures Mésencéphale dopamine cellulaire du nouveau-né contre les rongeurs

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

Recherche

Biologie

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.

Isolement d&#39;ARN à partir de synthèse d&#39;ADNc embryonnaires Zebrafish et pour analyser l&#39;expression génique

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.

Microdissection de tissus embryonnaires Zebrafish Eye

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.

Adultes et embryonnaires Culture squelettique Microexplant musculaire et isolement de cellules souches du muscle squelettique

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.

Dissection du cœur chez les larves, le poisson zèbre juvéniles et adultes, 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.

Immunocoloration des Disséqué coeur embryonnaire Zebrafish

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-infligé des blessures de poisson zèbre muscle squelettique embryonnaire

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.

Isolement et culture des fibres musculaires individuelles et de leurs cellules satellites du muscle squelettique adulte

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.

Imagerie et de reconstruction 3D de structures vasculaires cérébraux embryonnaires poisson zèbre

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.