Os autores gostariam de agradecer aos membros do laboratório Dowling (Aaron Reifler, Trent Waugh, Angela Busta, e William Telfer) que contribuíram para o desenvolvimento da técnica e da produção do manuscrito. Este trabalho foi financiado pelo Instituto Taubman, do Departamento de Pediatria da Universidade de Michigan, e em parte, de doações da Associação de Distrofia Muscular (JJD MDA186999) e os Institutos Nacionais de Saúde (JJD 1K08AR054835).

1. Preparação de poli-L-lisina revestido Lamelas (Tempo: 1 hora) Lamelas de revestimento permite a rápida colonização myofiber e aderência. Isto pode ser realizado durante o passo de dissociação do isolamento de células musculares (passo 2 abaixo). <ol><li> Cortar e colocar Parafilm na parte inferior de uma placa de Petri de 60 mm (qualquer marca). </li><li> Coloque deslizamentos de vidro da tampa do microscópio (12 mm de diâmetro) em Parafilm em um 60 milímetros de cultura de tecidos prato ou colocá-los individualmente em poços individuais de placas de 24 poços. </li></ol> Nota 1: Estas pequenas lamelas redondas ajudam a concentrar myofiber números e reduzir o excesso de uso de anticorpos. Nota 2: Outras lamelas tamanho vai funcionar, no entanto, os volumes de reagentes e embriões utilizados deverão ser ajustados em conformidade. <ol start="3"><li> Pipeta de 50-200 ul de solução de poli-L-lisina (0,01%) em cada tampa de vidro numa placa de Petri. </li><li> Permitir as lamelas de sentar-se pelo menos um hour a 37 ° C. Incubações mais longas não vai influenciar negativamente os resultados. </li><li> Remove-poli-L-lisina solução da tampa de vidro e lavar duas vezes com um mínimo de 100 l 1x PBS. </li><li> Permitir lamelas secar. </li><li> Lamelas está agora pronto para miofibras. </li></ol> Nota 3: A fim de garantir a esterilidade, o revestimento pode ser feito de um capô e em lamelas autoclavados. Nota 4: Mantenha a placa de Petri de 60 mm e Parafilm na parte inferior. Esta configuração será usada durante myofiber revestimento e imunomarcação (posteriores passos). Alternativa 1: Em vez de lamelas de revestimento imediatamente antes da utilização, um estoque lamela revestida pode ser utilizado. Um prato de 60 milímetros de Petri contendo um mínimo de 2 ml de poli-L-lisina pode ser armazenado a 4 ° C contendo numerosas lamelas. Com esta alternativa começar no passo 1.5 para processar as lamelas de miofibras. Alternativa 2: Também lamelas de casaco em poli-L-ornitina. To é mais trabalhosa, mas é útil para a cultura de longo prazo, porque lamelas revestidas com poli-L-ornitina pode ser tratado UV. Com o tratamento UV e técnica estéril cuidado myofibers ao vivo geralmente pode ser mantidas em cultura 4-7 dias. 2. A dissociação de embriões Zebrafish e chapeamento de fibras musculares (Tempo: 1 a 3 hr) Nota: o protocolo padrão se aplica melhor a 3 dpf (dias após a fertilização) Embriões. <ol><li> Transferência de embriões de peixes-zebra em um tubo de centrífuga de 1,5 ml padrão e remover o máximo de água de peixe possível. Tipicamente, pode ser utilizada 10-20 embriões por tubo, embora menos. Usando mais do que 20-25 embriões muitas vezes resulta numa preparação de densidade excessiva. <ol><li> Retirar córions antes da dissociação, utilizando manualmente # 5 fórceps. Alternativamente, a remoção do cório químico é conseguido através de um tratamento de 10-15 min com Pronase. Normalmente, a remoção prévia do córion só é necessária quando a confirmação prévia de um mutÉ necessário fenótipo formiga ou morfologia, ou quando usar estágios em que os embriões são naturalmente chocado de seu córion. </li></ol></li><li> Adicionar 900 ul de CO 2 meios independentes para o tubo contendo os embriões. </li><li> Adicionar 100 ul de colagenase tipo II, concentração final de 3,125 mg / ml (solução de colagenase a 31,25 mg / mL diluído em PBS 1x), para começar a dissociação. Colagenase IV também pode ser utilizado para a dissociação. </li><li> Rode embriões num agitador orbital, e tritura-se a cada 30 min em temperatura ambiente usando um P1000 Pipetman para a trituração. </li></ol> Nota 2: Acompanhar atentamente dissociação embrião; sobre ou sob a dissociação (especialmente over) são as razões mais comuns para a falha de protocolo. Os tempos para a digestão irá variar dependendo da intensidade de trituração, o número de embriões por tubo, e idade dos embriões. É também muitas vezes menos (em comparação com os tipos selvagens) para embriões de mutantes do músculo esquelético. Nota3: tempo de digestão médio por idade embrião: 1 dpf = 1 hora, 2 dpf = 1,5 hr, 3 dpf = 2 horas e 4 dpf = 2-2,5 horas. <ol start="5"><li> Pare de digestão quando há embriões inteiros são visíveis, mas pedaços sólidos ainda são visíveis - isto é essencial para prevenir overdigestion. </li><li> Tubos de centrífuga com embriões dissociadas em 0,8-2,3 g durante 3-5 minutos para as células de pelotas. </li><li> Remover o sobrenadante de células peletizadas e lavar 2x com CO 2 a mídia independente. </li><li> Adicionar fresco CO 2 mídia independente para ressuspender as células. 1 ml é normalmente usado para preparações de 10-20 embriões. O volume pode ser dimensionado com base no número de embriões. </li><li> Usando uma pipeta P1000, passar a suspensão através de um filtro de embriões de 70 mM. Isso ajuda a remover os resíduos indesejáveis ​​do prep. </li></ol> Nota 4: suspensão de embriões pode ser filtrado uma segunda vez, através de um filtro de 40 um para remover mais restos não desejados. A partir de uma dupla filtração, a recuperação é approximdiatamente 800 mL de um volume inicial de 1 ml. <ol start="10"><li> Adicionar cerca de 50-100 ul de suspensão de células musculares para cada lamela revestida com poli-L-lisina. </li></ol> Nota 5: Execute o passo 2.9 em placas de Petri com Parafilm no fundo, previamente preparado. O Parafilm mantém a suspensão de células musculares de correr as lamelas revestidas. Manter placas de Petri cobertas para evitar a evaporação. <ol start="11"><li> Permitir myofibers para resolver aproximadamente 1 hora para as lamelas revestidas à temperatura ambiente. </li></ol> Nota 6: fibras musculares começam a se estabelecer dentro de 5-10 min. No entanto, para uma boa pega myofiber, recomenda-se um mínimo de 1 hora (1-2 h). Permitindo myofibers que se contentar com mais tempo não irá prejudicar a preparação. Para incubações mais longos (incluindo durante a noite), os antibióticos podem ser adicionados aos meios de comunicação. Com antibióticos e técnica estéril culturas vivas geralmente pode ser mantida por 1-2 dias. <ol start="12"><li> Vivo myofibers pode ser observado neste ponto. Miofibras a partir de embriões 2 dpf e mais tarde será visto como células alongadas e estriadas (Figura 1). Neste ponto, myofibers agora está pronto para análise ao vivo ou por imunomarcação. </li></ol> Nota 7: O músculo esquelético a partir de 1 dpf embriões não placa fibras como alongados e claramente estriados. Em vez disso, grandes myoballs são visíveis. Além disso, durante a fase de granulação de pós de dissociação (passo 2.6), um dpf myoballs precisam de ser centrifugada durante um mínimo de 8 minutos a 5.000 xg, para atingir um sedimento. Para a análise de embriões, nesta fase, recomenda-se usar a linha transgénica que expressa EGFP especificamente no músculo esquelético 23. Isto irá permitir a identificação de células de origem muscular versus outras fontes. 3. Fluorescência imunocoloração de dissociadas Zebrafish miofibras (Tempo: cerca de 1 dia) 3.1. Imunomarcação <ol><li> Retire uma porçãomedia de fibras musculares aderidas à lamela revestido </li><li> Fix células usando 4% de paraformaldeído ou metanol. Para PFA, retire ½ o volume de mídia e substituir com a mesma quantidade de PFA. Correção para 10 min à temperatura ambiente, em seguida, remova volume total, substitua por 50-100 mL de PFA, e corrigi por mais 10 min. Para a fixação de álcool, metanol arrefecido em gelo ou acetona pode ser aplicado a 4 ° C durante 10 minutos ou 5 minutos, respectivamente. </li><li> Lavar miofibras, pelo menos, 3-5x com PBS 1x ou 1x PBS mais 0,1% de Tween. O volume médio de lavagem é 50-100 mL por lamela. </li><li> Adicionar solução para miofibras (trabalhando estoque) de bloqueio e incubar 20-60 minutos em temperatura ambiente. A solução de bloqueio irá variar dependendo dos anticorpos primários e secundários. Os dois mais comuns usadas no laboratório são: (1) PBS 1x, 2 mg / ml de BSA, 1% de soro de ovelha, 0,25% de Triton X-100 final e (2) 0,2% de Triton X-100, 2 mg / ml (0,2 % de BSA) e 5% de soro de ovelha / cabra. </li><li> Remova a solução de bloqueio e adicionar anticorpo primário diluído em qualquer bsolução ou PBS bloqueio. Incubar miofibras durante a noite a 4 ° C. Certifique-se de ambos os Parafilm ou embrulhe em Saran Wrap placa de Petri contendo as lamelas carregados de fibra muscular revestidas com anticorpos. Pequenos pedaços de toalha de papel humedecido pode ser adicionado para criar uma câmara humidificada. </li><li> Remover o anticorpo primário e lava-se a partir de lamelas lamelas de 3-5x durante 5 minutos com PBS ou solução de bloqueio. </li><li> Adicionar anticorpo secundário na concentração adequada diluída em 1x PBS ou solução de bloqueio durante 1-2 horas à temperatura ambiente. </li><li> Remover o anticorpo secundário e lava-miofibras 3-5x em PBS durante 5 minutos cada à temperatura ambiente. </li></ol> 3.2. Lamelas de montagem <ol start="9"><li> Aplicar 1-2 gotas de reagente antifade a uma lâmina de microscópio. </li><li> Cuidadosamente pick-up a lamela revestido e immunolabled usando uma pinça e lugar (myofibers para baixo) a lamela sobre o reagente antifade na lâmina de microscópio. </li></ol> Nota 1: Atenção para a orientação do clamela oated é crítica. <ol start="11"><li> Coloque 1-2 Kimwipes sobre uma superfície sólida duro. Inverter rapidamente o slide com as lamelas revestidas descansando sobre o reagente antifade e colocá-lo (lamela para baixo) na Kimwipes. </li><li> Aplicar uma leve pressão para os cantos da lâmina de microscópio para remover o excesso de Antifade e formar uma vedação estanque entre a lâmina e lamela </li><li> Permitir a Antifade restante secar durante 5-10 min à temperatura ambiente, em seguida, as fibras musculares estão prontos para a imagem (Figuras 2A e B) ou armazenar a 4 ° C. </li></ol> 4. Vivo celular Imagem de cálcio Usando GCaMP3 Experiências de células vivas pode ser realizada em miofibras antes da fixação (passo seguintes 2.10). O protocolo seguinte descreve a imagem ao vivo em miofibras expressando GCaMP3 24, um indicador de cálcio geneticamente codificado, expressa pelo específica zebrafish actina-α promotor músculo esquelético (pSKM) 23. Alternativamente,esta técnica pode ser facilmente adaptado para utilizar corantes indicadores de cálcio tais como o Fura-2. <ol><li> Injetar embriões com pSKM: GCaMP3 construir na fase 1-2 celular </li><li> Aos 3 dpf, coletar as larvas e preparam myofibers conforme descrito nas seções 1 e 2. Permitir miofibras-se aderir durante um período mínimo de 1 hora. Para esta técnica, que se preparam lamelas em pratos de 24 poços, é necessária. </li><li> Remova cuidadosamente qualquer excesso de mídia, e adicionar 300 ml de CO 2 mídia independente fresco à temperatura ambiente. </li><li> Observar células sob um microscópio invertido. GCaMP3 myofibers positivos deve ser visível sob fluorescência verde. </li><li> Configure câmera para a gravação, se desejar. </li><li> Prepare uma solução de cafeína 30 mM em CO 2 mídia independente. Para estimular as células, Pipeta suavemente 300 ml de solução de cafeína para o bem sob ampliação. Dentro 5-10 seg, GCaMP miofibras positivas deve responder a cafeína com um aumento rápido na fluorescência (Figura 3). Myofibers também podem contrair. De nota, a cafeína induz a liberação de cálcio do retículo sarcoplasmático das lojas 25. Outros agentes, tais como KCl 26 e ryanodine 4, pode ser usado para estudar liberação induzida de cálcio. </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>

Os autores declaram não haver conflito de interesses.

Zebrafish são um poderoso sistema modelo para o estudo de vertebrados desenvolvimento muscular e função in vivo 25,27,28. Eles também têm emergido como um recurso valioso para a modelagem de doenças musculares humanos 14,15,20,29. Enquanto grandes avanços têm sido tomadas para promover o uso e aplicação de peixe-zebra para o estudo da função muscular e doença muscular, há uma necessidade crítica constante para desenvolver ferramentas que permitem análise mais aprofundada qu...

O músculo esquelético é um tecido altamente especializado responsável por gerar as forças necessárias para a motilidade contráteis. A contração é iniciada através de um processo conhecido como excitação-contração (CE) acoplamento que converte os sinais elétricos para liberação de cálcio dos estoques intracelulares 1,2. Liberação de cálcio intracelular ativa o sarcômero para encurtar e gerar força. Os vários componentes específicos do maquinário molecular responsável por mediar a transmissão junção neuromuscular 3, acoplamento CE 4,5 e actina e miosina contrações dependentes 6 continuam a ser objecto de investigação em curso intenso. Além disso, as proteínas que estabilizam a membrana muscular durante 7,8 contracção e que medeiam a sinalização entre a fibra muscular e da matriz extracelular 7,9 foram identificados e estudados em grande detalhe. As mutações em vários genes importantes para a estrutura de um músculofunção nd foram identificados como causas de doenças do músculo esquelético humano ( <a href="http://www.musclegenetable.org/" target="_blank">http://www.musclegenetable.org/</a> ). Estas doenças, classificadas amplamente como miopatias e distrofias musculares esqueléticas com base em características clínicas e histopatológicas, estão associadas com fraqueza muscular, deficiência ao longo da vida, e 10,11 mortalidade precoce. O peixe-zebra tem provado ser um sistema excelente para modelar e estudar as doenças do músculo esquelético humanos 8,12,13. Ele tem sido empregado para validar as novas mutações genéticas 8, definir novos aspectos da doença fisiopatologia 14,15, e identificar novas abordagens terapêuticas 15,16. A potência do peixe-zebra para o estudo da doença muscular humana refere-se a um grande número de descendentes, o rápido desenvolvimento da estrutura muscular e na função, a clareza óptica do embrião do peixe-zebra, e a facilidade da manipulação genética e farmacológica da zebra desenvolvimentopeixe 17. Nós e outros 12,18,19 recentemente desenvolvida uma técnica simples para o isolamento rápido e eficiente de miofibras do peixe-zebra desenvolvimento. Esta metodologia permitiu o exame de miofibras em maior detalhe do que pode ser fornecida por análise de embrião inteiro. A técnica tem sido explorada para a caracterização da proteína de localização subcelular 20, bem como para a identificação de características histopatológicas importantes como parte de estudos de validação em modelos de doença recentemente desenvolvidos 21. Além disso, fibras musculares isolados podem ainda ser usado para geração de imagens ao vivo e para estudos eletrofisiológicos 22, técnicas que permitam o interrogatório dos aspectos-chave da função muscular. O protocolo específico para o isolamento de células musculares, junto com dois exemplos de experiências analíticas subseqüentes, é detalhado nas partes restantes deste 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

O peixe-zebra tem provado ser um sistema modelo valioso para explorar a função do músculo esquelético e para o estudo de doenças musculares humanas. Apesar das muitas vantagens oferecidas por análise in vivo de músculo esquelético, no peixe-zebra, visualizando o meio complexo e finamente estruturado proteína responsável pela função muscular, particularmente, em todo o embrião, pode ser problemático. Este impedimento resulta de o pequeno tamanho do músculo esquelético do peixe-zebra (60 mM) e o tamanho ainda menor do sarcómero. Aqui nós descrevemos e demonstrar um método simples e rápido para isolar miofibras esqueléticas a partir de embriões de peixes-zebra e larvas. Nós também incluímos protocolos que ilustram as técnicas de preparação de pós úteis para analisar a estrutura e função muscular. Especificamente, detalhar a localização imunocitoquímica posterior das proteínas do músculo esquelético e da análise qualitativa da liberação de cálcio estimulada através de imagem de cálcio de células vivas. No geral, este vídeoartigo fornece um método direto e eficaz para o isolamento e caracterização de fibras musculares esqueléticas de peixe-zebra, uma técnica que proporciona um canal para estudos subsequentes miríade de estrutura e função musculares.

Imunomarcação fluorescente de miofibras (Figura 2) As imagens mostram o esperado padrão de marcação fluorescente de miofibras imunocoradas após o isolamento bem sucedido e revestimento. As fibras têm sido marcada ou com anti-receptor de rianodina (1:100) (figura 2A) ou anti-α-actinina (1:100) (Figura 2B), anticorpos, e revelam imunocoloração da tríade e a banda Z respectivamente. Os anticorpos secundários utilizados fora...

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

Peixe-zebra são um sistema emergente para modelar doenças humanas do músculo esquelético. Nós descrevemos um método rápido e eficiente para isolar fibras musculares do músculo esquelético de peixe-zebra embrionário e larval. Este método conduz a uma preparação de alta densidade de células musculares adequado para o estudo da morfologia da fibra muscular esquelética única, a localização subcelular de proteínas, e da fisiologia do músculo.

Análise de Desenvolvimento Embrionário e larval Zebrafish esqueléticos miofibras de Preparações dissociadas

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 visualizações.  University of Michigan.  Peixe-zebra são um sistema emergente para modelar doenças humanas do músculo esquelético. Nós descrevemos um método rápido e eficiente para isolar fibras musculares do músculo esquelético de peixe-zebra embrionário e larval. Este método conduz a uma preparação de alta densidade de células musculares adequado para o estudo da morfologia da fibra muscular esquelética única, a localização subcelular de proteínas, e da fisiologia do músculo. 

Vídeo: 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.

Principal Dissociated Midbrain dopamina culturas de células de recém-nascidos de roedores

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

Biologia

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

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

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

Isolamento de RNA Síntese Zebrafish e cDNA embrionárias para Análise de Expressão Gênica

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.

Microdissecção de tecidos oculares Zebrafish embrionárias

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 e embrionárias Cultura Microexplant Músculo Esquelético e isolamento de células-tronco musculares

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.

Dissecção do coração em larval, Zebrafish Juvenil e Adulto, 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.

Imunomarcação de Dissecada Coração Zebrafish embrionárias

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 Lesão de Zebrafish muscular esquelético embrionário

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

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

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

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

Isolamento e Cultura de miofibras individuais e suas células satélites do músculo esquelético

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.

Imagem e Reconstrução 3D de Estruturas cerebrovasculares em 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 ...

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.