Agradecemos Bob Nowak (Andor Technology) para obter ajuda e aconselhamento técnico. SA-S. é apoiado por uma bolsa de Heisenberg da Deutsche Forschungsgemeinschaft (DFG). Este trabalho foi financiado pelo DFG concessão SE2016/7-1.

1. Rotular Células individuais Injectar embriões unicelulares palco com um plasmídeo que codifica GFP ou de qualquer proteína de fusão GFP sob o controlo de um promotor β-Actina. Durante o desenvolvimento, a GFP é então expressa de uma forma de mosaico. Aqui, utilizou-se o construto transgénico Tg [β-actina: α-actinina-GFP], o que coloca a proteína de fusão α-actinina-GFP sob o controlo do promotor da β-actina. 2. Incorporação de embrião <ol class="lalpha"><li> Coletar embriões zebrafish e manter em condições normais até 28 hpf (estadiamento de acordo com a 12). </li><li> Embriões Dechorionate sob o estereomicroscópio com uma pinça (Dumont # 5). </li><li> Prepara-se uma lâmina de microscópio com um anel de vaselina. </li><li> Preparar um 1% de baixo ponto de fusão de agarose / 10% de solução de tricaina com meio E3: fervura 100 mg de agarose em 10 ml de solução E3 para obter um líquido homogéneo e solução de agarose; aliquota e armazenarsolução de agarose não utilizado a -20 º C para posterior utilização. Para uso imediato, deixe a solução fervida esfriar e manter no máximo. 39 ° C. Adicionar tricaina solução stock para se obter uma concentração final de 10% tricaina. Ambos tricaina e agarose são necessárias para evitar movimentos embrionários. </li><li> Incorporação de um único embrião de 1% de ponto de fusão baixo a médio de agarose / 10% tricaina / E3 dentro do anel de vaselina, com o embrião fixa naturalmente no lado; suavemente cobrir com uma lamela apenas para manter o embrião no lugar e evitar um convexo superfície que iria quebrar a luz. Idealmente, o tecido do músculo embrionário que serão alvo deve tocar na lamela. </li></ol> 3. Embryo Laser ferimento <ol class="lalpha"><li> Prepare o laser Micropoint e do microscópio: <ol class="lroman"><li> Por favor, consulte o manual do laser Micropoint para instruções detalhadas sobre como configurar e utilizar o laser em conjunto com o microscópio. </li><li> Selecione o corretocorante para a geração de um feixe de laser 435 nm de comprimento de onda. </li><li> Ajustar a potência do laser usando o controle deslizante de atenuação. A potência do laser deve ser suficientemente forte para quebrar a superfície de vidro com um único impulso (por exemplo, o foco sobre a lamela de sua amostra para testar esta). Esta é a potência do laser necessária para a ablação de células. </li></ol></li><li> Concentre-se na região ou célula de interesse, utilizando o retículo instalado na ocular do microscópio. Use uma objetiva de 20x (40x permita danos mais preciso, mas muitas vezes a distância de trabalho não é suficiente para essa lente). </li></ol> Nota 1: O laser não só danificam as células no interior do plano de focagem, mas também as células acima e abaixo que se encontram dentro do raio laser. Certifique-se antes da experiência que o laser é perfeitamente configurado e ajustado para um foco máxima dentro do eixo z. É melhor para determinar a profundidade da lesão do laser antes de realizar o ensaio, a fim de saber qual células exatamente será ferido. <ol class="lalpha" start="3"><li> Defina-se a luz necessária para a visualização do experimento: brightfield para uso normal, ou fluorescência, se uma célula alvo marcada com fluorescência. </li><li> Enviar um impulso de luz laser para a região alvo, ou em vários pulsos, dependendo do grau desejado de lesão. </li></ol> 4. Análise de ferimento e Recuperação <ol class="lalpha"><li> Se desejado, o procedimento de lesão do laser pode ser gravado (por exemplo, utilizando a opção de aquisição fluxo do software Metamorph) 6. </li><li> Imediatamente após o ferimento, o embrião deve ser removido suavemente a partir da agarose com uma pinça e colocado de novo em água de ovo para a recuperação. </li></ol> Nota 2: A atividade muscular (natação e espasmos) é especialmente importante para a recuperação do músculo esquelético. Uma vez que o embrião não pode mover-se devido à exposição tricaina e a incorporação de agarose rígida, não é RECOMEnded para manter os embriões entre lâmina e lamínula por muito tempo. <ol class="lalpha" start="3"><li> Depois da recuperação, o embrião pode ser fixado e analisadas, conforme desejado pela luz, microscopia de fluorescência confocal ou conforme indicado. </li></ol>

<ol>
	<li>Charge, S. B., Rudnicki, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+regulation+of+muscle+regeneration.">Cellular and molecular regulation of muscle regeneration.</a> Physiol. Rev. 84, 209-238 (2004).</li><li>Steffen, L. S. <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+runzel+muscular+dystrophy+is+linked+to+the+titin+gene.">The zebrafish runzel muscular dystrophy is linked to the titin gene.</a> Dev. Biol. 309, 180-192 (2007).</li><li>Bassett, D., Currie, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+zebrafish+model+of+muscular+dystrophy.">Identification of a zebrafish model of muscular dystrophy.</a> Clin. Exp. Pharmacol. Physiol. 31, 537-540 (2004).</li><li>Kawahara, G., Serafini, P. R., Myers, J. A., Alexander, M. S., Kunkel, L. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+zebrafish+dysferlin+by+morpholino+knockdown.">Characterization of zebrafish dysferlin by morpholino knockdown.</a> Biochem. Biophys. Res. Commun. 413, 358-363 (2011).</li><li>Behra, M., Etard, C., Cousin, X., Str&#228;hle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+zebrafish+mutants+to+identify+secondary+target+effects+of+acetylcholine+esterase+inhibitors.">The use of zebrafish mutants to identify secondary target effects of acetylcholine esterase inhibitors.</a> Toxicol. Sci. 77, 325-333 (2004).</li><li>Otten, C., 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=Xirp+proteins+mark+injured+skeletal+muscle+in+zebrafish.">Xirp proteins mark injured skeletal muscle in zebrafish.</a> PLoS One. 7, e31041 (2012).</li><li>Roostalu, U., Str&#228;hle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+of+molecular+interactions+at+damaged+sarcolemma.">In vivo imaging of molecular interactions at damaged sarcolemma.</a> Dev. Cell. 22, 515-529 (2012).</li><li>Stickney, H. L., Barresi, M. J., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Somite+development+in+zebrafish.">Somite development in zebrafish.</a> Dev. Dyn. 219, 287-303 (2000).</li><li>Stellabotte, F., Dobbs-McAuliffe, B., Fernandez, D. A., Feng, X., Devoto, S. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+somite+cell+rearrangements+lead+to+distinct+waves+of+myotome+growth.">Dynamic somite cell rearrangements lead to distinct waves of myotome growth.</a> Development. 134, 1253-1257 (2007).</li><li>Choi, W. Y., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+regeneration.">Cardiac regeneration.</a> Curr. Top. Dev. Biol. 100, 319-344 (2012).</li><li>Stewart, S., Stankunas, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Limited+dedifferentiation+provides+replacement+tissue+during+zebrafish+fin+regeneration.">Limited dedifferentiation provides replacement tissue during zebrafish fin regeneration.</a> Dev. Biol. 365, 339-349 (2012).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. , 203-253 (1995).</li></ol>

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Laser mediada lesão é um método poderoso para infligir feridas de um tamanho desejado, a ablação de células, a fim de estudar a regeneração em condições controladas, o embrião do peixe-zebra. Notavelmente, as células podem ser direcionados precisamente (Figura 2), e tanto a área de lesão, assim como de temporização pode ser controlada. Subsequentemente, o local da lesão e os processos de regeneração são facilmente monitorizados, registada (Figura 3) e analisadas (Figura 1). No entanto, embora o laser é bem focado no x-e y-eixo, que não está completamente focada no eixo z. Isto pode provocar danos nas células situadas por baixo ou por cima das células de interesse. No entanto, esta abordagem abre um vasto campo de investigações em in vivo de reparação muscular.

<table border="1"><tbody><tr><td> Bloco de aquecimento </td><td></td><td></td><td></td></tr><tr><td> Par # 5 fórceps </td><td> Dumont </td><td></td><td></td></tr><tr><td> Lâminas de vidro </td><td> Menzel </td><td></td><td> 76 x 26 mm </td></tr><tr><td> Lamelas </td><td> Roth </td><td></td><td> 50 x 24 mm # 1 </td></tr><tr><td> Vaselina </td><td></td><td></td><td></td></tr><tr><td> Estereomicroscópio </td><td> Leica </td><td></td><td> MZFLIII </td></tr><tr><td> Micropoint a laser </td><td> Andor Tecnologia </td><td></td><td></td></tr><tr><td> Microscópio de fluorescência </td><td> Zeiss </td><td></td><td> Axioplan II </td></tr><tr><td> Metamorph software </td><td> Dispositivos Moleculares </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Reagentes <ul><li> Baixo ponto de fusão agarose (# 50081,Lonza) </li><li> Tricaina solução mãe: 400 mg tricaina (# A-5040, Sigma-Aldrich) / 100 ml de dH2O, pH 9,0 </li><li> Meio E3 (5 mM NaCl, 0,17 mM KCl, 0,33 mM CaCl2, 0,33 mM MgSO4) </li></ul></td></tr></tbody></table>

laser-inflicted injury of zebrafish embryonic skeletal muscle

Várias abordagens experimentais têm sido utilizados no mouse para induzir lesão muscular com o objetivo de estudar a regeneração muscular, incluindo injeções miotoxina (bupivacaína, cardiotoxina ou notexin), transplantes musculares (denervação desvascularização-regeneração induzida), exercício intensivo, mas também modelos murinos de distrofia muscular como o camundongo mdx (para uma revisão dessas abordagens ver 1). No peixe-zebra, abordagens genéticas incluem mutantes que exibem fenótipos de distrofia muscular (como runzel 2 ou sapje 3) e anti-sentido morpholinos oligonucleotídicas que bloqueiam a expressão de genes associados a distrofia 4. Além disso, as abordagens químicas também são possíveis, por exemplo, com a galantamina, um composto químico inibe a acetilcolinesterase, resultando assim em hipercontração, o que eventualmente leva a distrofia muscular 5. Abordagens entanto, genéticos e farmacológicos generally afectam todos os músculos no interior de um indivíduo, ao passo que a extensão dos ferimentos infligidos são fisicamente mais facilmente controlada espacialmente e temporalmente 1. Lesão física localizada permite a avaliação do músculo contralateral como controle interno. De facto, foi recentemente utilizado por laser de ablação celular mediada para estudar a regeneração do músculo esquelético no embrião do peixe-zebra 6, enquanto que um outro grupo recentemente relatada a utilização de um laser de dois fotões (822 nm) para danificar muito localmente na membrana plasmática de músculo de peixe zebra indivíduo embrionário células 7. Aqui, relatamos um método para a utilização do laser Micropoint (Andor Technology) por lesão do músculo esquelético de células no embrião do peixe-zebra. O laser Micropoint é um laser de alta energia, que é adequado para a ablação de células alvo num comprimento de onda de 435 nm. O laser é ligado a um microscópio (na nossa configuração, um microscópio óptico Zeiss partir) de tal modo que o microscópio pode ser utilizado para o mesmo tempo fou focalizar a luz de laser sobre a amostra e para a visualização dos efeitos do ferimento (campo claro ou de fluorescência). Os parâmetros para controlar o comprimento de onda de pulsos de laser incluem, a intensidade e o número de pulsos. Devido à sua transparência e desenvolvimento embrionário externo, o embrião do peixe-zebra é altamente favorável para ambos lesão induzida por laser e para estudar a recuperação posterior. Entre 1 e 2 dias após a fertilização, somíticos células do músculo esquelético progressivamente sofrerem maturação de anterior para posterior, devido à progressão da somitogénese do tronco para a cauda 8, 9. Nessas etapas, os embriões espontaneamente se contorcer e iniciar a natação. O peixe-zebra foi recentemente reconhecido como um importante organismo vertebrado modelo para o estudo da regeneração de tecidos, tal como vários tipos de tecidos (cardíaca, neuronal, vascular, etc) pode ser regenerado após a lesão no peixe-zebra adultos 10, 11.

Lesão mediada por laser foi realizada em imobilizadas um dia de idade, os embriões. Como mostrado na Figura 1, um impulso de laser que poucos podem gerar uma pequena ferida, facilmente reconhecível pelos danificadas, enroladas, Actina ricos miofibrilas, que normalmente são esticadas entre os limites somito. Um maior número de pulsos de laser vai, porém, resultar em um bloco somito maciçamente danificados, onde a maioria das miofibrilas são destruídos. No entanto, podemos observar que, literalmente, &quot;o tempo cura todas as feridas&quot;, com pequenas lesões de cura mais rápido do que os maiores. Após a recuperação, colorações de actina mostram que miofibrilas novamente abrangem o somito normalmente, no entanto, informações adicionais podem ser obtidas através da coloração de Xirp1. A proteína Xirp1 é um bom marcador para a detecção de lesões e em que tenha ocorrido recuperação, como está localizada ao longo ectopicamente recuperando miofibrilas dentro das células musculares anteriormente lesionados 6. Para alguns propósitos, é importante para determinar as células Exactly são feridos com o laser, por isso, pode ser útil para rotular geneticamente algumas células (por exemplo, por mosaically expressando uma proteína marcada com GFP), a fim de ser capaz de visualizar com precisão que é alvo das células e, em seguida, para avaliar se as suas células vizinhas têm foram também afectados pelo pulso de laser (Fig. 2). Após coloração lesão e da recuperação do anticorpo, que se torna possível encontrar e analisar a célula alvo novamente graças ao padrão de células marcadas mosaically rodeiam. Uma abordagem interessante é monitorar regularmente ao longo do tempo como a lesão de laser mediada afeta o músculo somítica. Para isto, o laser de lesão do embrião imobilizada pode ser gravado para ver os efeitos imediatos, por exemplo, dentro do primeiro minuto (Figura 3). Além disso, as gravações timelapse pode ser realizada com qualquer intervalo de tempo desejado (por exemplo, intervalo de 5 min), a fim de seguir de perto a forma como as células de reorganizarem torno da ferida. <img alt="Figura 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4351/4351fig1highres.jpg" src="/files/ftp_upload/4351/4351fig1.jpg" /> Figura 1. A gravidade da lesão infligida pode variar bastante, dependendo do número de impulsos de laser aplicado. Consequentemente, a recuperação da lesão irá proceder a ritmos de diferença, mas, eventualmente, a maioria das feridas são fechadas. Mostrámos recentemente que Xirp1 (verde) é um bom marcador para a regeneração do músculo, uma vez que é regulada positivamente após lesão e localiza a não-sarcomérica actina (vermelho) de miofibrilas danificadas a 32 hpf 6. <img alt="Figura 2" fo:content-width="6in" fo:src="/files/ftp_upload/4351/4351fig2highres.jpg" src="/files/ftp_upload/4351/4351fig2.jpg" /> Figura 2. Células marcadas pode ser alvo específico (modificado de Ref. 6). Cells foram marcadas pela expressão mosaico de Tg [β-actina: α-actinina-GFP] (verde ou preto e branco). Uma célula como aqui foi alvejado a 32 hpf e severamente danificada por pulsos de laser diversos, como indicado pelo parafuso vermelho. A seta branca indica a célula feridos; setas amarelas indicam outras células GFP-rotulados. A imagem da direita foi tomada após a recuperação e anticorpo coloração da mesma ferida. <img alt="Figura 3" fo:content-width="2in" fo:src="/files/ftp_upload/4351/4351fig3highres.jpg" src="/files/ftp_upload/4351/4351fig3.jpg" /> Figura 3. Aquisição transmissão ao vivo de lesão do laser revela como a lesão afeta o bloco somito inteiro. Montagem de fotos individuais registrados no Axioplan no modo de transmissão ao vivo (ou seja, cerca de 16 frames / seg) com DIC. A seta preta indica onde o laser atinge o músculo somíticos a 28 hpf. As setas vermelhas indicam a retração termina de uma danificadomiofibrila. Estas mudanças dinâmicas dentro de um bloco de somito ocorreu dentro de 2,5 segundos.

Watch this Scientific Journal Video about Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle at JoVE.com

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

O método aqui apresentado compreende a lesão precisa de embriões vivos zebrafish com pulsos de laser de alta potência e a subsequente análise das lesões e sua recuperação com o tempo. Também mostramos como geneticamente marcado individual ou grupos de células do músculo esquelético pode ser monitorado durante e após o laser danos leves induzido.

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 ...

laser-inflicted-injury-of-zebrafish-embryonic-skeletal-muscle

Nanyang Technological University

Research

JoVE Journal

Biology

10.4K visualizações.  Max Delbrück Center for Molecular Medicine. O método aqui apresentado compreende a lesão precisa de embriões vivos zebrafish com pulsos de laser de alta potência e a subsequente análise das lesões e sua recuperação com o tempo. Também mostramos como geneticamente marcado individual ou grupos de células do músculo esquelético pode ser monitorado durante e após o laser danos leves induzido.

Vídeo: Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

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 ...

Biologia

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 ...

Mitochondrial Isolation from Skeletal Muscle

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and regulate a number of signaling cascades2,3. Past and current researchers have isolated mitochondria from rat and mice tissues such as liver, brain and heart4,5. In recent years, many researchers have focused on studying mitochondrial function from skeletal muscles.
Here, we describe a method that we have used successfully for the isolation of mitochondria from skeletal muscles 6. Our procedure requires that all buffers and reagents are made fresh and need about 250-500 mg of skeletal muscle. We studied mitochondria isolated from rat and mouse gastrocnemius and diaphragm, and rat extraocular muscles. Mitochondrial protein concentration is measured with the Bradford assay. It is important that mitochondrial samples be kept ice-cold during preparation and that functional studies be performed within a relatively short time (~1 hr). Mitochondrial respiration is measured using polarography with a Clark-type electrode (Oxygraph system) at 37&deg;C7. Calibration of the oxygen electrode is a key step in this protocol and it must be performed daily. Isolated mitochondria (150 &mu;g) are added to 0.5 ml of experimental buffer (EB). State 2 respiration starts with addition of glutamate (5mM) and malate (2.5 mM). Then, adenosine diphosphate (ADP) (150 &mu;M) is added to start state 3. Oligomycin (1 &mu;M), an ATPase synthase blocker, is used to estimate state 4. Lastly, carbonyl cyanide p-[trifluoromethoxy]-phenyl-hydrazone (FCCP, 0.2 &mu;M) is added to measurestate 5, or uncoupled respiration 6. The respiratory control ratio (RCR), the ratio of state 3 to state 4, is calculated after each experiment. An RCR &ge;4 is considered as evidence of a viable mitochondria preparation.
In summary, we present a method for the isolation of viable mitochondria from skeletal muscles that can be used in biochemical (e.g., enzyme activity, immunodetection, proteomics) and functional studies (mitochondrial respiration).

This protocol describes a procedure to study the respiration of mitochondria isolated from skeletal muscles. This method was adapted from Scorrano et al. (2007). The mitochondrial isolation procedure requires about 2 hours. The mitochondrial respiration can be completed in about 1 hour.

Isolamento mitocondrial do músculo esquelético

Mitochondria are organelles controlling the life and death of the cell. They participate in key metabolic reactions, synthesize most of the ATP, and ...

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

Purificação de Progenitores do músculo esquelético

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

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 ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

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 ...

Assessment of Calcium Sparks in Intact Skeletal Muscle Fibers

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in the striated muscle fibers that appear as highly localized Ca2+ release events mediated by ryanodine receptor (RyR) Ca2+ release channels on the sarcoplasmic reticulum (SR) membrane. Proper assessment of muscle Ca2+ sparks could provide information on the intracellular Ca2+&#160;handling properties of healthy and diseased striated muscles. Although Ca2+ sparks events are commonly seen in resting cardiomyocytes, they are rarely observed in resting skeletal muscle fibers; thus there is a need for methods to generate and analyze sparks in skeletal muscle fibers.
Detailed here is an experimental protocol for measuring Ca2+ sparks in isolated flexor digitorm brevis (FDB) muscle fibers using fluorescent Ca2+ indictors and laser scanning confocal microscopy. In this approach, isolated FDB fibers are exposed to transient hypoosmotic stress followed by a return to isotonic physiological solution. Under these conditions, a robust Ca2+ sparks response is detected adjacent to the sarcolemmal membrane in young healthy FDB muscle fibers. Altered Ca2+ sparks response is detected in dystrophic or aged skeletal muscle fibers. This approach has recently demonstrated that membrane-delimited signaling involving cross-talk between inositol (1,4,5)-triphosphate receptor (IP3R) and RyR contributes to Ca2+ spark activation in skeletal muscle. In summary, our studies using osmotic stress induced Ca2+ sparks showed that this intracellular response reflects a muscle signaling mechanism in physiology and aging/disease states, including mouse models of muscle dystrophy (mdx mice) or amyotrophic lateral sclerosis (ALS model).

Described here is a method to directly measure calcium sparks, the elementary units of Ca2+ release from sarcoplasmic reticulum&#160;in intact skeletal muscle fibers. This method utilizes osmotic-stress-mediated triggering of Ca2+ release from ryanodine receptor in isolated muscle fibers. The dynamics and homeostatic capacity of intracellular Ca2+ signaling can be employed to assess muscle function in health and disease.

Avaliação do cálcio Faíscas intactas fibras musculares esqueléticas

Maintaining homeostatic Ca2+ signaling is a fundamental physiological process in living cells. Ca2+ sparks are the elementary units of Ca2+ signaling in ...

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.

Zebrafish (Danio rerio) have become a particularly effective tool for modeling human diseases affecting skeletal muscle, including muscular ...

Nephrotoxin Microinjection in Zebrafish to Model Acute Kidney Injury

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid decline in renal function and development of the clinical syndrome known as acute kidney injury (AKI). Pharmacological agents used to treat medical circumstances ranging from bacterial infection to cancer, when administered individually or in combination with other drugs, can initiate AKI. Zebrafish are a useful animal model to study the chemical effects on renal function in vivo, as they form an embryonic kidney comprised of nephron functional units that are conserved with higher vertebrates, including humans. Further, zebrafish can be utilized to perform genetic and chemical screens, which provide opportunities to elucidate the cellular and molecular facets of AKI and develop therapeutic strategies such as the identification of nephroprotective molecules. Here, we demonstrate how microinjection into the zebrafish embryo can be utilized as a paradigm for nephrotoxin studies.

Renal injuries incurred from nephrotoxins, which include drugs ranging from antibiotics to chemotherapeutics, can result in complex disorders whose pathogenesis remains incompletely understood. This protocol demonstrates how zebrafish can be used for disease modeling of these conditions, which can be applied to the identification of renoprotective measures.

Nefrotoxina microinjeção no peixe-zebra ao modelo de lesão renal aguda

The kidneys are susceptible to harm from exposure to chemicals they filter from the bloodstream. This can lead to organ injury associated with a rapid ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

Isolamento e Caracterização de Exossomos de Fibroblastos de Músculo Esquelético

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the ...