Ringraziamo Trent Waugh per la sua assistenza tecnica. Riconosciamo inoltre il Dipartimento di Pediatria presso l&#39;Hospital for Sick Children e cura distrofia muscolare congenita (CMD) per il loro generoso finanziamento per questo progetto.

1. Preparazione di piastre di agar iniezione (Tempo: 45 min) <ol><li> Far bollire 2% al 3% agarosio nei media E3 e lasciare raffreddare la soluzione un po &#39;sul banco. Nota: Il numero di piatti di iniezione viene preparato determina la quantità di agarosio richiesta. Ciascuna piastra di iniezione deve circa 35 ml della soluzione di agarosio. </li><li> Dopo l&#39;ebollizione, lasciare l&#39;agarosio raffreddare fino al raggiungimento della temperatura desiderata (ad es., 45 ° C) secondo le istruzioni del produttore dello stampaggio ad iniezione. </li><li> Versare circa 35 ml di agarosio raffreddati in un piatto 100 mm. </li><li> Mettere una delle estremità del stampo iniezione preferito in soluzione, quindi copritelo resto di muffe su soluzione di agarosio (questo contribuirà a ridurre la presenza di bolle d&#39;aria). </li><li> Lasciare la soluzione di agarosio solidificare sia a RT o 4 ° C per circa 30 min. </li><li> Utilizzare una spatola per separare una estremità dello stampo dal agarosio solido. Rimuovere lentamente il resto del m<html> vecchio. </li></ol> 2. Preparazione di Evans blu tipo (EBD) Iniezione Mix (Tempo: 30 min) <ol><li> Fare un magazzino 1% di EBD in soluzione di 1X Ringer (155 mM NaCl, 5 mM KCl; 2mM CaCl 2; 1 mM MgCl 2; 2 mM Na 2 HPO 4; HEPES 10 mm; glucosio 10 mm; pH a 7.2), che possono essere conservati a temperatura ambiente. </li><li> Ottenere una soluzione stock di isotiocianato di fluorescina (FITC) -dextran MW 10.000 kDa a 25 mg / ml in soluzione di Ringer 1X e conservare a -20 ° C. </li><li> Preparare mix iniezione diluendo EBD al 0,1% direttamente in soluzione madre di FITC-destrano magazzino (cioè, per un volume finale di lavoro di 100 microlitri. Miscelare 10 ml di 1% DEB in 90 ml di destrano FITC magazzino). </li><li> Mescolare bene iniezione vortice (dovrebbe diventare verde), e tenere fuori dalla luce diretta avvolgendo tubo mix di iniezione in un foglio di alluminio. </li></ol> 3. EBD iniezione Preparazione (Tempo: circa 30 min) ent &quot;&gt; Nota: Protocollo funziona meglio con larve da 3-7 giorni dopo la fecondazione (DPF). <ol><li> Piastra iniezione Preriscaldare a RT. </li><li> Istituito apparato iniezione disponendo micromanipolatore su lastra di metallo e stare accanto al microscopio in uso per l&#39;iniezione. Accendere l&#39;aria guidato controllore microiniezione. Nota: Il sistema di iniezione preferito varierà da laboratorio e non dovrebbe cambiare risultato dell&#39;analisi. </li><li> Torna riempire ago per iniezione con circa 2-4 ml di mix EBD. </li><li> Calibrare volume di iniezione a circa 5 nl di mix EBD. Nota: la calibrazione Volume di iniezione dipenderà dal metodo di calibrazione. Una iniezione pistone guidato può direttamente essere impostato su un dato volume di iniezione che iniettori pressione del gas dovranno il volume di iniezione tarato tramite il volume di bolo con l&#39;uso di un micrometro. </li><li> Piastra iniezione bagnato con la soluzione di Ringer 1X e rimuovere l&#39;eccesso dai pozzi. </li><li> Pre-treat larve con 0,04% di etile 3 amminobenzoato methanesulfonate salt (tricaine) diluito in soluzione di Ringer 1X per immobilizzare larve prima dell&#39;inizio dell&#39;iniezione. Nota: Assicurare le larve sono completamente immotile è importante in quanto l&#39;iniezione corretta è difficile con qualsiasi movimento residuo. </li><li> Posizionare larve anestetizzato nei pozzetti delle piastre di agar iniezione utilizzando una pipetta di vetro. Assicurarsi che le larve sono completamente all&#39;interno del pozzo, che giace su un fianco. Nota: Il numero di larve per bene è fino allo sperimentatore. </li><li> Dopo le larve sono messi nei pozzetti, rimuovere la soluzione in eccesso di Ringer per ridurre al minimo il movimento larve all&#39;interno del pozzo. Lascia un importo residuo di soluzione in modo che le larve non si disidrata. </li></ol> 4. Iniezione pericardico di Zebrafish larve con EBD (Tempo: A seconda del numero di larve per via parenterale, stimato 1-3 ore) <ol><li> Posizionare la piastra di iniezione contenente le larve in un ambito dissezione dove verranno eseguite le iniezioni. </li><li> Posizionare l&#39;ago pipetta di iniezione contenenteil EBD mix su un larve di pesce zebra. </li><li> Riposizionare la piastra di iniezione facendolo ruotare in modo che l&#39;ago iniezione è vicino il cuore del larve e circa 45 ° ventralmente dal asse anteriore-posteriore. </li><li> Inserire l&#39;ago di iniezione nella vena cardinale comune (CCV) nella regione della vena in corrispondenza della porzione anteriore del tuorlo cui la vena è inizialmente ruota nella direzione dorsale (Figura 1). Nota: Un ingrandimento di fino a 40x può essere utile per vedere chiaramente il CCV. </li><li> Iniettare 5 nl del mix di EBD e tenere ago per iniezione in posizione per 5-8 secondi per ridurre al minimo la perdita immediata di mix EBD. Nota: Una buona iniezione avrà colorante colorazione visto nelle camere cardiache (Figura 1). Se mix EBD non si osserva nel cuore, poi iniettando un ulteriore 5 nl dell&#39;impasto EBD può essere sufficiente per indurre l&#39;assorbimento di colorante. In alternativa, l&#39;embrione può essere scartato. Nota: In alcune situazioni, il cuore potrebbe smettere di battere. Se questo occurs, continuerà a monitorare le larve per 20-40 secondi. Tipicamente, il cuore riprende battere come il colorante si muove attraverso il sistema circolatorio. </li><li> Passare al prossimo larve e ripetere. </li><li> Identificare embrioni iniettati con successo osservando la presenza di FITC-destrano nella vascolatura immediatamente dopo l&#39;iniezione (Figura 2). </li></ol> 5. Incubazione e EBD Assorbimento (Tempo: 4-6 ore) <ol><li> Dopo il numero desiderato di larve vengono iniettati, ritorno iniettato larve soluzione 1X Ringer senza tricaine a 100 mm piatti. </li><li> Tenere piatti avvolti in un foglio di alluminio. Nota: Mantenere larve iniettato nel buio aumenta in modo significativo i tassi di sopravvivenza e garantisce la massima coerenza nella potenza del segnale. Involucro in alluminio è particolarmente importante per il periodo di tempo in cui le larve sono al di fuori del termostato. </li><li> Lasciare che le larve di incubare a 28,5 ° C per 4-6 ore per garantire EBD sufficienti assorbimento. </li></ol&gt; 6. Visualizzazione dei DEB nel muscolo (Time: A seconda del numero di larve iniezione e del tipo di microscopio, previsto per 0,5-3 ore) <ol><li> Prima di imaging, anestetizzare le larve con 0,04% tricaine per impedire il movimento. </li><li> Vista sotto larve fluorescenza rossa per determinare se EBD assorbimento si verifica nel muscolo scheletrico (Figura 3). </li></ol>

<ol>
	<li>Verma, S., Anziska, Y., Cracco, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Review+of+Duchenne+muscular+dystrophy+(DMD)+for+the+pediatricians+in+the+community.">Review of Duchenne muscular dystrophy (DMD) for the pediatricians in the community.</a> Clin Pediatr (Phila. 49, 1011-1017 (2010).</li><li>Gibbs, E. M., Horstick, E. J., Dowling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Swimming+into+prominence:+the+zebrafish+as+a+valuable+tool+for+studying+human+myopathies+and+muscular+dystrophies).">Swimming into prominence: the zebrafish as a valuable tool for studying human myopathies and muscular dystrophies).</a> FEBS J. 280, 4187-4197 (2013).</li><li>Lancet, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , 381-845 (2013).</li><li>Lapidos, K. A., Kakkar, R., McNally, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+dystrophin+glycoprotein+complex:+signaling+strength+and+integrity+for+the+sarcolemma.">The dystrophin glycoprotein complex: signaling strength and integrity for the sarcolemma.</a> Circ Res. 94, 1023-1031 (2004).</li><li>Campbell, K. P., Kahl, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Association+of+dystrophin+and+an+integral+membrane+glycoprotein.">Association of dystrophin and an integral membrane glycoprotein.</a> Nature. 338, 259-262 (1989).</li><li>Cohn, R. D., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+basis+of+muscular+dystrophies.">Molecular basis of muscular dystrophies.</a> Muscle Nerve. 23, 1456-1471 (2000).</li><li>Yoshida, M., Ozawa, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycoprotein+complex+anchoring+dystrophin+to+sarcolemma.">Glycoprotein complex anchoring dystrophin to sarcolemma.</a> J Biochem. 108, 748-752 (1990).</li><li>Straub, V., Rafael, J. A., Chamberlain, J. S., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+muscular+dystrophy+show+different+patterns+of+sarcolemmal+disruption.">Animal models for muscular dystrophy show different patterns of sarcolemmal disruption.</a> J Cell Biol. 139, 375-385 (1997).</li><li>Matsuda, R., Nishikawa, A., Tanaka, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visualization+of+dystrophic+muscle+fibers+in+mdx+mouse+by+vital+staining+with+Evans+blue:+evidence+of+apoptosis+in+dystrophin-deficient+muscle.">Visualization of dystrophic muscle fibers in mdx mouse by vital staining with Evans blue: evidence of apoptosis in dystrophin-deficient muscle.</a> J Biochem. 118, 959-964 (1995).</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>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>Kawahara, G., 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=Drug+screening+in+a+zebrafish+model+of+Duchenne+muscular+dystrophy.">Drug screening in a zebrafish model of Duchenne muscular dystrophy.</a> Proc Natl Acad Sci U S A. 108, 5331-5336 (2011).</li><li>Guyon, J. R., 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=Modeling+human+muscle+disease+in+zebrafish.">Modeling human muscle disease in zebrafish.</a> Biochim Biophys Acta. 1772, 205-215 (2007).</li><li>Cavanna, J. S., 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=Molecular+and+genetic+mapping+of+the+mouse+mdx+locus.">Molecular and genetic mapping of the mouse mdx locus.</a> Genomics. 3, 337-341 (1988).</li><li>Bassett, D. I., 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=Dystrophin+is+required+for+the+formation+of+stable+muscle+attachments+in+the+zebrafish+embryo.">Dystrophin is required for the formation of stable muscle attachments in the zebrafish embryo.</a> Development. 130, 5851-5860 (2003).</li><li>Horstick, E. 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=Stac3+is+a+component+of+the+excitation-contraction+coupling+machinery+and+mutated+in+Native+American+myopathy.">Stac3 is a component of the excitation-contraction coupling machinery and mutated in Native American myopathy.</a> Nat Commun. 4, (1952).</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>Telfer, W. R., Nelson, D. D., Waugh, T., Brooks, S. V., Dowling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neb:+a+zebrafish+model+of+nemaline+myopathy+due+to+nebulin+mutation.">Neb: a zebrafish model of nemaline myopathy due to nebulin mutation.</a> Dis Model Mech. 5, 389-396 (2012).</li><li>Dowling, J. 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=Oxidative+stress+and+successful+antioxidant+treatment+in+models+of+RYR1-related+myopathy.">Oxidative stress and successful antioxidant treatment in models of RYR1-related myopathy.</a> Brain. 135, 1115-1127 (2012).</li><li>Detrich, H. W., Westerfield 3rd, ., M, L. I., Zon, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Overview of the Zebrafish system. Methods Cell Biol. 59, 3-10 (1999).</li><li>Whitfield, T. T., 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+development+of+the+zebrafish+inner+ear+and+lateral.">Mutations affecting development of the zebrafish inner ear and lateral.</a> , 123-241 (1996).</li><li>Kawahara, G., Guyon, J. R., Nakamura, Y., 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=Zebrafish+models+for+human+FKRP+muscular+dystrophies.">Zebrafish models for human FKRP muscular dystrophies.</a> Hum Mol Genet. 19, 623-633 (2010).</li><li>Baraban, S. C., Dinday, M. T., Hortopan, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+screening+in+Scn1a+zebrafish+mutant+identifies+clemizole+as+a+potential+Dravet+syndrome+treatment.">Drug screening in Scn1a zebrafish mutant identifies clemizole as a potential Dravet syndrome treatment.</a> Nat Commun. 4, (2013).</li><li>Waugh, T. 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=Fluoxetine+prevents+dystrophic+changes+in+a+zebrafish+model+of+Duchenne+muscular+dystrophy.">Fluoxetine prevents dystrophic changes in a zebrafish model of Duchenne muscular dystrophy.</a> Hum Mol Genet. 23 (17), 4651-4662 .</li><li>Horstick, E. J., Gibbs, E. M., Li, X., Davidson, A. E., Dowling, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+embryonic+and+larval+zebrafish+skeletal+myofibers+from+dissociated+preparations.">Analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations.</a> J Vis Exp. 50259, (2013).</li><li>Smith, L. L., Beggs, A. H., Gupta, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+skeletal+muscle+defects+in+larval+zebrafish+by+birefringence+and+touch-evoke+escape+response+assays.">Analysis of skeletal muscle defects in larval zebrafish by birefringence and touch-evoke escape response assays.</a> J Vis Exp. 50925, (2013).</li><li>Hamer, P. W., McGeachie, J. M., Davies, M. J., Grounds, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evans+Blue+Dye+as+an+in+vivo.+marker+of+myofibre+damage:+optimising+parameters+for+detecting+initial+myofibre+membrane+permeability.">Evans Blue Dye as an in vivo. marker of myofibre damage: optimising parameters for detecting initial myofibre membrane permeability.</a> J Anat. 200, 69-79 (2002).</li><li>Guyon, J. R., 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=Genetic+isolation+and+characterization+of+a+splicing+mutant+of+zebrafish+dystrophin.">Genetic isolation and characterization of a splicing mutant of zebrafish dystrophin.</a> Hum Mol Genet. 18, 202-211 (2009).</li><li>Santoriello, C., Zon, L. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hooked!+Modeling+human+disease+in+zebrafish.">Hooked! Modeling human disease in zebrafish.</a> J Clin Invest. 122, 2337-2343 (2012).</li><li>Majczenko, K., 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=Dominant+Mutation+of+CCDC78+in+a+Unique+Congenital+Myopathy+with+Prominent+Internal+Nuclei+and+Atypical+Cores.">Dominant Mutation of CCDC78 in a Unique Congenital Myopathy with Prominent Internal Nuclei and Atypical Cores.</a> Am J Hum. , (2012).</li><li>Wooddell, C. I., 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=Use+of+Evans+blue+dye+to+compare+limb+muscles+in+exercised+young+and+old+mdx+mice.">Use of Evans blue dye to compare limb muscles in exercised young and old mdx mice.</a> Muscle Nerve. 41, 487-499 (2010).</li><li>Hall, T. 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=The+zebrafish+candyfloss+mutant+implicates+extracellular+matrix+adhesion+failure+in+laminin+alpha2-deficient+congenital+muscular+dystrophy.">The zebrafish candyfloss mutant implicates extracellular matrix adhesion failure in laminin alpha2-deficient congenital muscular dystrophy.</a> Proc Natl Acad Sci U S A. 104, 7092-7097 (2007).</li></ol>

Gli autori non hanno interessi finanziari in competizione o altri conflitti di interesse.

Zebrafish stanno emergendo come un potente strumento per lo studio delle malattie neuromuscolari 2,29 malattia. Ad oggi, il sistema di zebrafish è stato utilizzato per convalidare nuovi muscoli che causano malattie mutazioni 16,17,30, chiarire nuovi meccanismi patogenetici 18, e di identificare potenziali nuovi farmaci terapeutici 12,24. Questi sforzi collettivi hanno stabilito l&#39;utilità del zebrafish per modellare malattie neuromuscolari umane. Tuttavia, nonostante i progressi compiuti con modelli zebrafish e mammiferi, ci sono limitate opzioni di trattamento per i pazienti all&#39;interno del vasto spettro di condizioni neuromuscolari. Pertanto, esiste una forte domanda di sviluppo di terapie per questo gruppo di malattie devastanti. Parallelamente questa richiesta di terapie è la corrispondente necessità di continua innovazione sperimentale, così come l&#39;analisi rigorosa per verificare nuovi modelli animali e putativi strategie terapeutiche. Analisi EBD è comunemente utilizzato in modelli murini pertessuto studio e danno cellulare nel cervello, cuore, e scheletrico 27,31 muscolare. In particolare, EBD sono ampiamente usate in modelli murini di vari sottotipi distrofia muscolare di mostrare la gravità del muscolo membrana instabilità e danneggiare 8. L&#39;uso di EBD per rivelare danni membrana muscolare è un parametro di supporto che stabilisce analogie del modello animale per lo stato di malattia umana 9. Il potere di EBD nel topo ha condotto numerosi laboratori, compreso il nostro, per sviluppare e applicare EBD ai modelli zebrafish di malattie neuromuscolari. A causa della applicabilità di analisi EBD, questa tecnica è attivamente in corso di attuazione per corroborare modelli zebrafish allo stato di malattia umana 11,15,22,24,32. Larve con membrane muscolari danneggiate avrà EBD assorbimento e fluorescenza, pertanto rosso all&#39;interno fibre muscolari. Fluorescenza osservato nello spazio inter-fibra, ma non all&#39;interno singole fibre muscolari possono anche essere informativo di fibre si staccano dalla membrana basale in tegli assenza di danno alla membrana, fornendo utili dettagli diagnostico. Analisi EBD ha un potenziale di applicazione al di là di validazione dei modelli animali. Gli sforzi del nostro laboratorio hanno recentemente dimostrato che l&#39;analisi EBD è utile nel convalidare potenzialmente nuovi farmaci terapeutici 24. Determinare se i potenziali trattamenti terapeutici ridurre o abolire EBD assorbimento in modelli di malattia neuromuscolare può significare rilevanti azione terapeutica 8. Questo tipo di analisi può aiutare a stabilire il meccanismo (s) di terapie ed espande l&#39;applicazione dell&#39;analisi EBD. Come per molte tecniche, analisi EBD l&#39;ha diversi avvertimenti da osservare durante la progettazione e la pratica sperimentale. Ad esempio, può essere difficile identificare la causa del CCV ispessimento del tessuto con l&#39;età. Inoltre, è facile danneggiare larve in preparazione prima e durante l&#39;iniezione pericardico, riducendo il numero sperimentali e aumentando la necessità di preparare un gran numero di larve.Inoltre, il danno fisico fatto per le larve durante la movimentazione e l&#39;iniezione può causare falsi positivi come muscolo danneggiato può richiedere fino EBD. Per superare alcuni di questi ostacoli, abbiamo descritto una strategia di co-iniezione in questo articolo video che permette l&#39;identificazione semplice e affidabile di larve con successo infusione colorante immediatamente dopo l&#39;iniezione e prima della successiva analisi. La FITC-destrano controlli co-iniezione per iniezione di successo consentendo conferma dei DEB nel sistema vascolare prima del suo assorbimento da parte delle fibre muscolari. Questo può essere particolarmente utile come EBD fluorescenza diventa altamente diffuso in larve dopo alcune ore se non raccolti nelle fibre muscolari; come tale, può essere difficile da rilevare. Inoltre, manca il CCV e iniettando EBD nel tuorlo o corpo cavità può, dopo incubazione, comportare diffusa fluorescenza rossa simile a controllare gli embrioni, ma con la minore probabilità di assorbimento da fibre muscolari danneggiate. Collettivamente, questi grottaats suggeriscono iniezione EBD richiede pazienza e pratica al fine di ottenere risultati coerenti e affidabili. In tutto, si descrive un metodo pratico e semplice per eseguire analisi EBD su larve di zebrafish. Ad oggi, l&#39;uso di zebrafish come sistema modello, in particolare come un modello di malattia umana, è in rapida espansione. Questa espansione è in parte dovuto al continuo sviluppo e modifica di tecniche sperimentali che migliorano sugli attuali vantaggi del sistema zebrafish. La tecnica di iniezione EBD fornisce uno strumento aggiuntivo e potente per l&#39;arsenale di un ricercatore per la validazione e lo studio di modelli di malattie muscolo zebrafish. L&#39;attuazione in corso e la modifica di questa tecnica ha il potenziale per aiutare a scoprire nuove strategie terapeutiche, nonché i meccanismi che provocano la malattia.

Muscular dystrophies constitute a group of prevalent and heterogeneous human muscle diseases with specific histopathological features1,2. Symptoms typically associated with this devastating group of diseases include muscle weakness, muscle degeneration, loss of motility, and early mortality1,3. The primary pathomechanisms of muscular dystrophies are the loss of proteins that stabilize the sarcolemma, anchor transmembrane complexes, and mediate cell signaling across the membrane4-6. For example, complete loss of the protein dystrophin, a primary scaffold protein of the dystrophin-glycoprotein complex, results in destabilization of the muscle membrane in Duchenne muscular dystrophy7. Due to the fact that most muscular dystrophies result from mutations in proteins that participate in the link between the extracellular matrix and the sarcolemmal cytoskeleton, a common observation at the cellular level is the loss of sacrolemmal integrity8,9. This understanding of the primary pathomechanism(s) associated with muscular dystrophies is the product of numerous years of research employing animal model systems2,10-15. However, despite advances in the field, there are still limited therapeutic options for treatment or management of the range of dystrophy subtypes. This limitation of viable therapies is due to several key factors: 1) the difficulty of gene therapy strategies, 2) a high frequency of de-novo disease cases and the corresponding lack of translatable animal models, and 3) the lack of rigorous strategies to test the physiological consequences of putative therapeutic agents with clear and reproducible outcome measures.
To overcome some of these limitations, numerous labs including our own are employing zebrafish as a system to model and study human neuromuscular diseases2. To date, zebrafish have proven a valuable tool in disease research. The zebrafish model has been used to identify and validate novel human disease causing mutations16,17, elucidate uncharacterized disease causing mechanisms17,18, and identify novel therapeutic strategies12,19. These advances were made, in part, by the canonical strengths of the zebrafish system such as their optical clarity, ease of genetic manipulation, and ability to breed in large numbers20. Zebrafish have additionally proven amendable to large-scale drug screens21, a valuable method for the identification of novel therapeutics22-24. Regarding muscle disease research, these strengths are complemented by the ability to isolate single zebrafish skeletal muscle fibers via dissociation25 and by the ability to examine myofiber organization in vivo using the optical phenomenon called birefringence26, which collectively allows for rapid determination of macroscopic muscle integrity. Regardless of these available utilities, further tool development is continuously required to advance investigation.
We, and others, have adapted a protocol for EBD injection and analysis in the zebrafish model. EBD is a vital, cell permeable dye that is taken up by damaged, degenerating, or apoptotic cells and then visualized under fluorescence27. To date, EBD analysis has extensively been used to analyze muscle membrane integrity in mouse models of skeletal muscle and heart diseases8,9,27. However, in mammalian preparations, harvested muscle typically requires laborious sectioning or dissection prior to analysis. In zebrafish, direct analysis is possible in high numbers using live and intact animals. In this video article, we will demonstrate the methodology to perform EBD injection and analysis in live zebrafish larvae, with representative images of EBD uptake in the zebrafish dystrophy mutant line sapje15,28. Furthermore, we present a co-injection strategy that allows for increased quantification of EBD preparations.

<table border="0" cellpadding="0" cellspacing="0" width="801">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Fluorescein isothiocyanate-dextran MW 10,000</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">FD10S</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Evan&#39;s Blue Dye</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">E2129</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Ethyl 3-aminobenzoate methanesulfonate salt</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">A5040</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">100 mm Petri dish</td>
			<td style="width:173px;">Fischerbrand</td>
			<td style="width:140px;">FB0875712</td>
			<td>Injection mold base</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:321px;">Thin wall glass capillaries</td>
			<td style="width:173px;">World Precision Instruments</td>
			<td style="width:140px;">TW100F-4</td>
			<td>For Injection needle</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Agarose</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">AGA001</td>
			<td>Injection mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Microinjection mold</td>
			<td style="width:173px;">Adaptive Science Tools</td>
			<td style="width:140px;">TU-1</td>
			<td>Injection mold</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sodium chloride</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">SOD001</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Potassium chloride</td>
			<td style="width:173px;">Bioshop</td>
			<td style="width:140px;">POC888</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Magnessium chloride hexahydrate</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">M2670</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Sodium phosphate monobasic monohydrate</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">S9638</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">HEPES</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">H4034</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Glucose</td>
			<td style="width:173px;">BioBasic</td>
			<td style="width:140px;">GB0219</td>
			<td>Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:321px;">Calcium chloride</td>
			<td style="width:173px;">Sigma</td>
			<td style="width:140px;">C1061</td>
			<td>Ringer&#39;s solution</td>
		</tr>
	</tbody>
</table>

analysis of zebrafish larvae skeletal muscle integrity with evans blue dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

Il mix di iniezione EBD è stato iniettato nel CCV di mutanti omozigoti sapje e wild-type fratelli a 3 dpf. Iniezioni che riempiva camere cardiache (Figura 1B) sono stati poi analizzati per iniezione di successo visualizzando FITC-destrano nella vascolatura sotto fluorescenza verde (Figura 2). Dopo un periodo di incubazione di 4 ore, EBD assorbimento è stata esaminata a livello somite usando la microscopia a fluorescenza. Fratelli di tipo selvatico esposti alcuna fluorescenza EBD entro eventuali fibre muscolari visibili (Figura 3a), mentre i mutanti omozigoti sapje mostrato EBD assorbimento, che indica un danno alla membrana muscolo 15 (Figura 3B). <img alt="Figura 1" src="/files/ftp_upload/53183/53183fig1.jpg" /> Figura 1. Iniettare EBD mix iniezione nella vena cardinale comune (CCV) di un Embry zebrafisho. (A) uninjected embrione. Freccia indica il luogo ideale per l&#39;iniezione CCV. (B) iniezione di successo nella CCV. Il colorante entra nelle camere cardiache (freccia) e inizia ad essere pompato attraverso il sistema vascolare. (C) Un&#39;iniezione CCV senza successo si tradurrà in alcuni o tutti del colorante entrare nel sacco vitellino dell&#39;embrione (freccia). <a href="https://www.jove.com/files/ftp_upload/53183/53183fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53183/53183fig2.jpg" /> Figura 2. Gli embrioni possono essere ordinati per la iniezione di successo osservando distribuzione FITC-destrano sotto fluorescenza verde per tutto il sistema vascolare immediatamente dopo l&#39;iniezione e prima EBD assorbimento.Cliccate qui per vedere una versione più grande di questa figura. <img alt="Figura 3" src="/files/ftp_upload/53183/53183fig3.jpg" /> Figura 3: EBD sarà ripreso da fibre con membrane danneggiate fratelli (A) di tipo selvatico che mostrano alcuna fluorescenza EBD nelle fibre muscolari.. (B) Sapje ​​mutante omozigote con fluorescenza EBD entro fibre muscolari multipli (frecce). Tutte le larve sono stati iniettati con il mix iniezione EBD e analizzato dopo un periodo di incubazione di 4 ore a 3 dpf. Fratelli e mutanti sono stati ordinati per muscolo distacco fibra prima dell&#39;iniezione CCV. <a href="https://www.jove.com/files/ftp_upload/53183/53183fig3large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye at JoVE.com

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Analisi delle larve Zebrafish muscolo scheletrico Integrità con Blu di Evans Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

analysis-zebrafish-larvae-skeletal-muscle-integrity-with-evans-blue

Research

JoVE Journal

Developmental Biology

9.0K Views.  The Hospital for Sick Children. In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Video: Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Catturare Tissue Repair in Zebrafish Larve con Time-lapse Brightfield stereomicroscopia

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Ricerca

Biologia dello sviluppo

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

Induzione di acuta muscolo scheletrico Rigenerazione da cardiotossina Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Microstructured Devices for Optimized Microinjection and Imaging of Zebrafish Larvae

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning fundamental developmental biology through to large-scale genetic and chemical screens. However, many experiments, especially those related to infection and xenograft models, rely on microinjection and imaging of embryos and larvae, which are laborious techniques that require skill and expertise. To improve the precision and throughput of current microinjection techniques, we developed a series of microstructured devices to orient and stabilize zebrafish embryos at 2 days post fertilization (dpf) in ventral, dorsal, or lateral orientation prior to the procedure. To aid in the imaging of embryos, we also designed a simple device with channels that orient 4 zebrafish laterally in parallel against a glass cover slip. Together, the tools that we present here demonstrate the effectiveness of photolithographic approaches to generate useful devices for the optimization of zebrafish techniques.

Microinjection of zebrafish embryos and larvae is a crucial but challenging technique used in many zebrafish models. Here, we present a range of microscale tools to aid in the stabilization and orientation of zebrafish for both microinjection and imaging.

Dispositivi microstrutturati Microinjection ottimizzata e Imaging delle larve di Zebrafish

Zebrafish have emerged as a powerful model of various human diseases and a useful tool for an increasing range of experimental studies, spanning ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

Applicazione di stimolazione cronica per lo studio del ratto indotta da attività contrattile del muscolo scheletrico fenotipica adattamenti

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Identificazione delle cellule satelliti del muscolo scheletrico dall'immunofluorescenza con Pax7 e Laminin anticorpi

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

Isolamento, cultura, caratterizzazione e differenziazione delle cellule del progenitore muscolare umano dalla procedura di biopsia muscolare scheletrica

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

Esecuzione di Xenografi muscolari scheletrici umani in topi immunodeficienti

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Isolamento e differenziazione dei mioblasti primari dagli esopanti muscolari scheletrici del topo

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...

Whole Mount Immunohistochemistry in Zebrafish Embryos and Larvae

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. Although many immunohistochemistry protocols have been optimized for mammalian tissue and tissue sections, these protocols often require modification and optimization for non-mammalian model organisms. Zebrafish are increasingly used as a model system in basic, biomedical, and translational research to investigate the molecular, genetic, and cell biological mechanisms of developmental processes. Zebrafish offer many advantages as a model system but also require modified techniques for optimal protein detection. Here, we provide our protocol for whole-mount fluorescence immunohistochemistry in zebrafish embryos and larvae. This protocol additionally describes several different mounting strategies that can be employed and an overview of the advantages and disadvantages each strategy provides. We also describe modifications to this protocol to allow detection of chromogenic substrates in whole mount tissue and fluorescence detection in sectioned larval tissue. This protocol is broadly applicable to the study of many developmental stages and embryonic structures.

Here, we present a protocol for fluorescent antibody-mediated detection of proteins in whole preparations of zebrafish embryos and larvae.

Intero Monte Immunohistochimica in Embrioni e Larve di pesce zebra

Immunohistochemistry is a widely used technique to explore protein expression and localization during both normal developmental and disease states. ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Esame della rigenerazione muscolare nei modelli zebrafish di malattia muscolare

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...