Los autores desean agradecer a los miembros del laboratorio de Dowling (Aaron Reifler, Trent Waugh, Angela Busta, y William Telfer) que contribuyeron al desarrollo de la técnica y para la producción del manuscrito. Este trabajo fue financiado por el Instituto Taubman, el Departamento de Pediatría de la Universidad de Michigan, y en parte de subvenciones de la Asociación de Distrofia Muscular (JJD MDA186999) y los Institutos Nacionales de Salud (JJD 1K08AR054835).

1. Preparación de poli-L-lisina recubierto cubreobjetos (Tiempo: 1 hr) Cubreobjetos de recubrimiento permite una rápida sedimentación de miofibras y adhesión. Esto se puede realizar durante la etapa de disociación del aislamiento de miofibras (paso 2 a continuación). <ol><li> Cortar y colocar Parafilm en la parte inferior de una placa de 60 mm de Petri (de cualquier marca). </li><li> Coloca los resbalones de la cubierta de vidrio de microscopio (12 mm de diámetro) en Parafilm en un 60 mm placa de cultivo de tejidos o colocarlos individualmente en pocillos individuales de placas de 24 pocillos. </li></ol> Nota 1: Estos pequeños cubreobjetos redondos ayudan a concentrarse miofibras números y reducir el uso de exceso de anticuerpo. Nota 2: Otros cubreobjetos tamaño trabajarán, sin embargo, tendrán que ajustarse en consecuencia los volúmenes de los reactivos y de los embriones utilizados. <ol start="3"><li> Pipeta de 50-200 l de solución de poli-L-lisina (0,01%) en cada vidrio de cubierta en la placa de Petri. </li><li> Deje que los portaobjetos para sentarse por lo menos una hour a 37 ° C. Incubaciones más largas no influirán negativamente en los resultados. </li><li> Retire la solución de poli-L-lisina de la cubierta de vidrio y se lava dos veces con un mínimo de 100 l de PBS 1x. </li><li> Permitir cubreobjetos se sequen. </li><li> Cubreobjetos ya están listos para las miofibrillas. </li></ol> Nota 3: Para asegurar la esterilidad, de recubrimiento se puede hacer en una campana y en cubreobjetos tratados en autoclave. Nota 4: Mantener el 60 mm placa de Petri con Parafilm en la parte inferior. Esta configuración se utilizará durante miofibra galvanoplastia y immunolabeling (posteriores pasos). Alternativa 1: En lugar de cubreobjetos de revestimiento inmediatamente antes del uso, un cubreobjetos de papel estucado se puede utilizar. Un plato de 60 mm de Petri que contiene un mínimo de 2 ml de poli-L-lisina se puede almacenar a 4 ° C que contiene numerosos cubreobjetos. Con esta alternativa comience en el paso 1.5 para procesar los cubreobjetos de las miofibrillas. Alternativa 2: También cubreobjetos capa de poli-L-ornitina. Tla suya es más mano de obra, pero es útil para el cultivo a largo plazo porque cubreobjetos recubiertos con poli-L-ornitina puede ser tratado UV. Con el tratamiento UV y una técnica estéril cuidado miofibras en vivo típicamente se pueden mantener en cultivo de 4-7 días. 2. Disociación de embriones de pez cebra y Plating de miofibras (Tiempo: 1 a 3 horas) Nota: el protocolo estándar se aplica mejor a 3 dpf (días después de la fecundación) embriones. <ol><li> Transferencia de embriones de pez cebra en un tubo de centrífuga de 1,5 ml estándar y eliminar la mayor cantidad de agua el exceso de pescado como sea posible. Por lo general, se pueden utilizar 10 a 20 embriones por tubo, aunque menos. El uso de más de 20-25 embriones a menudo resulta en una preparación de densidad excesiva. <ol><li> Eliminar chorions antes de la disociación, usando manualmente # 5 fórceps. Alternativamente, la eliminación corion química se consigue con un 10-15 min tratamiento con pronasa. Por lo general, sólo es necesaria la eliminación previa del corion cuando la confirmación previa de un mutSe requiere fenotipo hormiga o morfología, o cuando se utilizan etapas donde los embriones son, naturalmente, nacido de su corion. </li></ol></li><li> Añadir 900 l de CO 2 medios de comunicación independientes para el tubo que contiene los embriones. </li><li> Añadir 100 l de colagenasa de tipo II, una concentración final de 3,125 mg / ml (solución madre colagenasa a 31,25 mg / ml diluida en 1X PBS) para comenzar la disociación. La colagenasa IV también se puede utilizar para la disociación. </li><li> Girar embriones en un agitador orbital, y se tritura cada 30 min a temperatura ambiente usando un P1000 Pipetman para la trituración. </li></ol> Nota 2: Es importante vigilar cuidadosamente la disociación de embriones; sobre o debajo de la disociación (sobre todo mayores) son las razones más comunes para el fracaso del protocolo. Los tiempos para la digestión pueden variar dependiendo de la intensidad de la trituración, el número de embriones por tubo, y la edad de los embriones. También es a menudo menos (en comparación con los tipos salvajes) para embriones de los mutantes del músculo esquelético. Nota3: tiempo de digestión promedio por edad del embrión: 1 dpf = 1 hora, 2 dpf = 1,5 horas, 3 dpf = 2 h, y 4 dpf = 2-2,5 horas. <ol start="5"><li> Detener la digestión cuando no hay embriones enteros son visibles, todavía trozos sólidos siguen siendo visibles - esto es esencial para prevenir overdigestion. </li><li> Tubos centrífuga con embriones disociadas a 0,8-2,3 g durante 3-5 minutos a las células de pellets. </li><li> Aspirar el sobrenadante de células sedimentadas y lavar 2 veces con CO 2 medios de comunicación independientes. </li><li> Añadir CO fresco 2 medios de comunicación independientes para volver a suspender las células. 1 ml se utiliza normalmente para preparaciones de 10-20 embriones. El volumen se puede escalar basada en el número de embriones. </li><li> Con una pipeta P1000, pasar la suspensión del embrión a través de un filtro de 70 micras. Esto ayuda a eliminar los residuos no deseados de la preparación. </li></ol> Nota 4: suspensión de embriones se puede filtrar una segunda vez a través de un filtro de 40 micras para eliminar adicionalmente los desechos no deseados. A partir de una doble filtración, la recuperación es aproximadam ente 800 l de un volumen inicial de 1 ml. <ol start="10"><li> Añadir aproximadamente 50-100 l de suspensión de miofibras a cada cubreobjetos recubiertos con poli-L-lisina. </li></ol> Nota 5: Realice el paso 2.9 en las placas de Petri con Parafilm en la parte inferior, previamente preparado. El Parafilm mantiene la suspensión miofibra escurra los cubreobjetos recubiertos. Mantenga placas Petri cubiertas para evitar la evaporación. <ol start="11"><li> Permitir miofibras se asienten aproximadamente 1 hr en los cubreobjetos recubiertos a temperatura ambiente. </li></ol> Nota 6: miofibras comenzarán a instalarse dentro de 5-10 min. Sin embargo, para un buen agarre miofibra, se recomienda un mínimo de 1 hora (1-2 horas). Permitir que las miofibrillas se asienten durante más tiempo no dañará la preparación. Para incubaciones más largas (incluyendo durante la noche), los antibióticos pueden ser añadidos a los medios de comunicación. Con antibióticos y técnica estéril cultivos vivos normalmente se pueden mantener durante 1-2 días. <ol start="12"><li> Vivo myofibers se puede observar en este punto. Miofibrillas de embriones 2 DPF y más tarde se verá como células estriadas y alargadas (Figura 1). En este punto, las miofibras ya están listas para análisis en vivo o para immunolabeling. </li></ol> Nota 7: El músculo esquelético de 1 fdd embriones no platear fibras alargadas y claramente estriadas. En cambio, las grandes myoballs son visibles. Además, durante la fase de granulación mensaje de disociación (paso 2.6), 1 dpf myoballs necesitan ser centrifuga durante un mínimo de 8 minutos a 5000 xg para obtener un pellet. Para el análisis de los embriones en esta etapa, se recomienda utilizar la línea transgénica que expresa EGFP específicamente en el músculo esquelético 23. Esto permitirá la identificación de las células de origen muscular en comparación con otras fuentes. 3. Fluorescencia inmunotinción de disociadas de pez cebra miofibras (Tiempo: aproximadamente 1 día) 3.1. Immunolabeling <ol><li> Retire una parte demedios de miofibras adheridas a la cubreobjetos recubierto </li><li> Fijar las células utilizando paraformaldehído al 4% o metanol. Para PFA, retire ½ el volumen de los medios de comunicación y reemplazar con la misma cantidad de PFA. Fijar durante 10 minutos a temperatura ambiente, a continuación, extraiga el volumen total, reemplace con 50-100 l de PFA, y fijar, por una suma adicional de 10 min. Para la fijación de alcohol, metanol enfriado con hielo o acetona se puede aplicar a 4 º C durante 10 min o 5 min, respectivamente. </li><li> Lávese las miofibrillas al menos 3 a 5 veces con 1x PBS o 1x PBS más 0,1% de Tween. El volumen medio de lavado es de 50 a 100 l por cubreobjetos. </li><li> Añadir la solución de bloqueo a las miofibrillas (solución de trabajo) e incubar 20-60 minutos a temperatura ambiente. El bloqueo de solución variará dependiendo de los anticuerpos primarios y secundarios. Los dos más comunes utilizados en el laboratorio son (1) 1x PBS, 2 mg / ml de BSA, 1% de suero de ovejas, 0,25% de Triton X-100 final y (2) 0,2% de Triton X-100, 2 mg / ml (0,2 % de BSA) y 5% de suero de oveja / cabra. </li><li> Eliminar la solución de bloqueo y añadir anticuerpo primario diluido en cualquiera de bsolución de PBS o de bloqueo. Incubar miofibras la noche a 4 ° C. Asegúrese de que sea Parafilm o envuelva en Saran Wrap la placa de Petri que contiene los cubreobjetos miofibras cargada recubiertas con anticuerpos. Pequeños trozos de toalla de papel humedecida se pueden añadir para crear una cámara humidificada. </li><li> Eliminar el anticuerpo primario de cubreobjetos y lavar cubreobjetos 3-5x por 5 min con PBS o solución de bloqueo. </li><li> Añadir anticuerpo secundario a una concentración apropiada diluida en 1X PBS o solución de bloqueo durante 1-2 horas a TA. </li><li> Se quita el anticuerpo secundario y lavar miofibras 3-5x en PBS durante 5 min cada uno a temperatura ambiente. </li></ol> 3.2. Cubreobjetos de montaje <ol start="9"><li> Aplicar 1-2 gotas de antifade reactivo a un portaobjetos de microscopio. </li><li> Pick-up con cuidado el cubreobjetos recubierto y immunolabled utilizando fórceps y lugar (miofibrillas hacia abajo) el cubreobjetos sobre la antifade reactivo en el portaobjetos de un microscopio. </li></ol> Nota 1: La atención a la orientación de la ccubreobjetos oated es crítica. <ol start="11"><li> Coloque 1-2 Kimwipes sobre una superficie sólida duro. Invertir rápidamente la diapositiva con los cubreobjetos recubiertos de descanso en la antifade reactivo y colocarlo (cubreobjetos hacia abajo) en la Kimwipes. </li><li> Aplique una leve presión sobre las esquinas del portaobjetos para retirar el exceso Antifade y formar un sello hermético entre el portaobjetos y cubreobjetos </li><li> Permitir la Antifade restante secar durante 5-10 minutos a temperatura ambiente, y luego las fibras musculares están listos para la imagen (Figuras 2 A y B) o se almacenan a 4 ° C. </li></ol> 4. Celular en vivo Imagen de calcio Usando GCaMP3 Experimentos con células vivas pueden ser realizados en fibras musculares antes de la fijación (siguientes paso 2,10). El siguiente protocolo describe imágenes en vivo en miofibras que expresan GCaMP3 24, un indicador de calcio codificado genéticamente, expresado por el promotor pez cebra específica del músculo esquelético-α actina (pSKM) 23. Alternativamente,esta técnica se puede adaptar fácilmente para utilizar colorantes indicadores de calcio tales como Fura-2. <ol><li> Inyectar embriones con pSKM: GCaMP3 construir en la etapa de 1-2 células </li><li> A las 3 dpf, recoger larvas y preparar las miofibrillas como se describe en las secciones 1 y 2. Permitir miofibras que se adhieran durante un mínimo de 1 hora. Para esta técnica, se requiere la preparación de cubreobjetos en placas de 24 pocillos. </li><li> Retire con cuidado el exceso de medios de comunicación, y añadir 300 l de fresco CO 2 medios de comunicación independientes a TA. </li><li> Observar las células bajo un microscopio invertido. Miofibras GCaMP3 positivos deben ser visibles bajo fluorescencia verde. </li><li> Preparar la cámara para la grabación, si se desea. </li><li> Prepare una solución de cafeína 30 mM en CO 2 medios de comunicación independientes. Para estimular las células, pipetear suavemente 300 l de solución de cafeína en el pozo con una lupa. Dentro de 5-10 segundos, miofibras GCaMP positivos deben responder a la cafeína con un rápido aumento de la fluorescencia (Figura 3). Myofibres también pueden contraer. Es de destacar que la cafeína induce la liberación de calcio de los almacenes de retículo sarcoplásmico 25. Otros agentes, como KCl 26 y rianodina 4, se pueden utilizar para estudiar la liberación de calcio inducible. </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>

Los autores declaran no tener conflictos de intereses.

El pez cebra es un modelo de sistema de vertebrados de gran alcance para el estudio de desarrollo de los músculos y la función en vivo 25,27,28. Ellos también se han convertido en un activo valioso para el modelado de enfermedades musculares humanas 14,15,20,29. Aunque se han dado grandes pasos para avanzar en el uso y aplicación de pez cebra para el estudio de la función muscular y la enfermedad del músculo, hay una necesidad crítica constante para desarrollar herramientas que perm...

El músculo esquelético es un tejido altamente especializado responsable de generar las fuerzas contráctiles necesarias para la motilidad. La contracción se inicia a través de un proceso conocido como excitación-contracción (CE) de acoplamiento que convierte las señales eléctricas a la liberación de calcio desde las reservas intracelulares 1,2. La liberación de calcio intracelular activa el sarcómero se acorte y generar fuerza. Los numerosos componentes específicos de la maquinaria molecular responsable de mediar la transmisión unión neuromuscular 3, acoplamiento CE 4,5, y la actina-miosina contracciones dependientes 6 continúan siendo objeto de una intensa investigación en curso. Además, las proteínas que estabilizan la membrana muscular durante 7,8 contracción y que median la señalización entre la miofibras y la matriz extracelular 7,9 se han identificado y estudiado en gran detalle. Las mutaciones en un número de genes importantes para la estructura de un músculoND función se han identificado como causas de enfermedades del músculo esquelético humanos ( <a href="http://www.musclegenetable.org/" target="_blank">http://www.musclegenetable.org/</a> ). Estas enfermedades, que se clasifican en términos generales como miopatías esqueléticas y distrofias musculares basados ​​en las características clínicas e histopatológicas, se asocian a debilidad muscular, incapacidad permanente y 10,11 mortalidad temprana. El pez cebra ha demostrado ser un sistema excelente para modelar y estudiar las enfermedades del músculo esquelético humanos 8,12,13. Se ha empleado para validar nuevas mutaciones del gen 8, definir nuevos aspectos de la fisiopatología de la enfermedad 14,15, e identificar nuevos enfoques terapéuticos 15,16. El poder del pez cebra para el estudio de la enfermedad de músculo humano se refiere a la gran cantidad de el rápido desarrollo de la estructura muscular y la función, la claridad óptica del embrión de pez cebra, y la facilidad de manipulación genética y farmacológica de la cebra el desarrollo de la descendencia,pescado 17. Nosotros y otros 12,18,19 hemos desarrollado recientemente una técnica simple para el aislamiento rápido y eficiente de las miofibras del pez cebra en desarrollo. Esta metodología ha permitido el examen de las miofibras en mayor detalle que puede ser proporcionada por el análisis de embriones conjunto. La técnica se ha explotado para la caracterización de la proteína de localización subcelular 20, así como para la identificación de importantes características histopatológicas como parte de los estudios de validación en modelos de enfermedad de nuevo desarrollo 21. Además, las miofibras aisladas, además, se pueden usar para formación de imágenes en vivo y para los estudios electrofisiológicos 22, técnicas que permiten la interrogación de los aspectos clave de la función muscular. El protocolo específico para el aislamiento de miofibras, junto con dos ejemplos de experimentos analíticos posteriores, se detalla en las partes restantes de este manuscrito.

<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

El pez cebra ha demostrado ser un sistema modelo valioso para la exploración de la función del músculo esquelético y para el estudio de enfermedades musculares humanas. A pesar de las muchas ventajas que ofrece el análisis in vivo de los músculos esqueléticos en el pez cebra, visualizando el complejo y finamente estructurada entorno proteína responsable de la función muscular, especialmente en los embriones en su conjunto, puede ser problemático. Este obstáculo se deriva del pequeño tamaño del músculo esquelético pez cebra (60 m) y el tamaño aún más pequeño del sarcómero. Aquí describimos y demostrar un método sencillo y rápido para el aislamiento de miofibras esqueléticas a partir de embriones de pez cebra y larvas. También se incluyen los protocolos que muestran técnicas de correos de preparación útiles para el análisis de la estructura y la función muscular. Concretamente, se detalla la localización inmunocitoquímica posterior de las proteínas del músculo esquelético y el análisis cualitativo de la liberación de calcio estimulado a través de imágenes de calcio de células vivas. En general, este videoartículo proporciona un método sencillo y eficiente para el aislamiento y caracterización de miofibras esqueléticas de pez cebra, una técnica que proporciona un conducto para estudios posteriores miríada de estructura y la función muscular.

Inmunomarcaje fluorescente de miofibras (Figura 2) Las imágenes que muestra un patrón marcado fluorescente que se espera de las miofibrillas inmunotiñeron después del aislamiento y de las planchas de éxito. Las fibras musculares se han etiquetado con cualquier anti-receptor de rianodina (1:100) (Figura 2A) o anti-α-actinina (1:100) (Figura 2B) anticuerpos, y revelar la inmunotinción de la tríada y la banda Z, respectivamente...

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

El pez cebra es un sistema emergente para modelar enfermedades humanas del músculo esquelético. Se describe un método rápido y eficaz para aislar las miofibrillas del músculo esquelético de pez cebra embrionario y larval. Este método proporciona una preparación de alta densidad miofibra adecuada para el estudio de la morfología sola fibra muscular esquelética, localización subcelular de proteínas, y la fisiología muscular.

Análisis de embriones y larvas de pez cebra esqueléticas miofibras de Preparativos disociadas

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 vistas.  University of Michigan.  El pez cebra es un sistema emergente para modelar enfermedades humanas del músculo esquelético. Se describe un método rápido y eficaz para aislar las miofibrillas del músculo esquelético de pez cebra embrionario y larval. Este método proporciona una preparación de alta densidad miofibra adecuada para el estudio de la morfología sola fibra muscular esquelética, localización subcelular de proteínas, y la fisiología muscular. 

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 disociada Mesencéfalo culturas dopamina celulares de roedores recién nacidos

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

Investigación

Biología

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.

Aislamiento del ARN de síntesis embrionarias pez cebra y el cDNA de análisis de expresión génica

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.

Microdisección de los tejidos del ojo de pez cebra embrionarias

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.

Adultas y embrionarias del músculo esquelético Cultura Microexplant y aislamiento de células madre de músculo esquelético

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.

La disección del corazón en las larvas, juveniles y adultos de pez cebra, 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.

Inmunotinción de Disección del corazón embrionario de pez cebra

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-infligido lesiones de músculo esquelético embrionario de pez cebra

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.

Aislamiento y cultivo de fibras individuales y sus células satélite del músculo esquelético adulto

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.

Imágenes y Reconstrucción 3D de Estructuras Embrionarias cerebrovasculares en pez cebra

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.