Biz teknik yardım ve danışma için Bob Nowak (Andor Teknolojisi) teşekkür ederim. SA-S. Deutsche Forschungsgemeinschaft (DFG) bir Heisenberg dostluk tarafından desteklenmektedir. Bu eser hibe SE2016/7-1 DFG tarafından desteklenmiştir.

1. Tek Hücreler Etiketleme Bir β-aktin promotörü kontrolü altında bir plasmid kodlama GFP ya da GFP füzyon proteini ile tek-hücreli embriyoların enjekte edilir. Gelişimi sırasında, GFP sonra bir mozaik bir şekilde ifade edilir. Burada kullanılan transgenik yapı Tg [β-aktin: α-aktinin-GFP] olup β-aktin promotörü kontrolü altında α-aktinin-GFP füzyon proteini yerleştirir. 2. Embriyo Katıştırma <ol class="lalpha"><li> Zebrafish embriyolar toplayın ve 28 hpf (evreleme 12 göre) kadar normal koşullar altında korur. </li><li> Forseps (Dumont # 5) ile birlikte bir stereomikroskop altında Dechorionate embriyolar. </li><li> Bir vazelin halka ile bir mikroskop slayt hazırlayın. </li><li> Bir% 1 düşük erime noktalı agaroz / E3 orta ile% 10 tricaine çözelti hazırlayın: bir sıvı ve homojen bir çözelti elde etmek için agaroz 10 ml E3 çözelti içinde kaynamaya 100 mg, agaroz, tablet ve mağazadaha sonra kullanmak için -20 ° C &#39;de kullanılmayan agaroz çözelti. Acil kullanım için, haşlanmış çözüm soğumasını ve max muhafaza edelim. 39 ° C. % 10 tricaine bir nihai konsantrasyon elde etmek için tricaine stok solüsyonu eklenir. Tricaine ve agaroz Hem embriyonik hareketlerin önlenmesi için gereklidir. </li><li> Embriyo tarafında doğal döşeme ile,% 1 vazelin halka içinde düşük erime agaroz / 10% tricaine / E3 ortamda tek bir embriyo Embed; nazikçe yerine embriyo korumak için ve bir dışbükey önlemek için bir lamel ile kaplayın ışığı kıracak yüzey. İdeal hedeflenecektir embriyonik kas dokusu lamel temas etmelidir. </li></ol> 3. Embriyo Lazer-yaralama <ol class="lalpha"><li> Micropoint lazer ve mikroskop hazırlayın: <ol class="lroman"><li> Mikroskop ile birlikte kurmak ve lazer nasıl kullanılacağı hakkında ayrıntılı talimatlar için Micropoint lazer kılavuzuna bakın. </li><li> Doğru SeçinBir 435 nm dalga boylu lazer ışını üretimi için boya. </li><li> Zayıflama kaydırıcısını kullanarak lazer gücünü ayarlayın. Lazer gücü, tek bir darbe (bu test için numune lamel örneğin odaklama) ile cam yüzey kırmak için yeterince güçlü olmalıdır. Bu, hücre ablasyon için gerekli lazer güçtür. </li></ol></li><li> Mikroskobun oküler yüklü retikül kullanarak ilgi bölgesi veya hücre odaklanın. 20x objektif (40x daha kesin hasar izin verir, ancak genellikle çalışma mesafesi bu lens için yeterli değildir) kullanın. </li></ol> Not 1: lazer sadece odaklama düzlemi içindeki hücrelere zarar değil, aynı zamanda yukarıda hücreleri ve lazer ışını dahilinde altında değildir. Lazer, optimum kurmak ve z ekseni içinde bir maksimal odak için ayarlanmış olduğundan emin denemeden önce emin olun. Bunu bilmek için deney yapmadan önce lazer yaralanma derinliğini belirlemek için en iyi olan cepİşte aynen yaralı olacaktır. <ol class="lalpha" start="3"><li> Bir floresan etiketli hücre hedefleme eğer normal kullanım veya floresan için aydınlık: Deneme görüntülenmesi için gerekli ışığı ayarlayın. </li><li> Yaralanma istenilen derecesine bağlı olarak, hedef bölge, ya da birkaç puls için lazer bir ışık gönder. </li></ol> 4. Yaralama ve Kurtarma Analizi <ol class="lalpha"><li> Eğer istenirse, lazer yaralanma prosedür canlı (örn. Metamorph yazılım akışı elde etme seçenek kullanılarak). 6 kaydedilebilir </li><li> Hemen yaralama sonra, embriyo forseps ile agaroz gelen yavaşça kaldırılır ve kurtarma için yumurta suya geri konulmalıdır. </li></ol> Not 2: Atletik aktivite (yüzme ve seğirmesi) iskelet kas kurtarma için özellikle önemlidir. Embriyo tricaine pozlama ve sert agaroz gömme nedeniyle hareket edemez bu yana, recomme değildirçok uzun süre slayt ve lamel arasındaki embriyo korumak için nded. <ol class="lalpha" start="3"><li> Belirtildiği gibi kurtarma sonra, embriyo ışık-, floresan veya konfokal mikroskopi ile bağlanmış ve istenildiği gibi analiz edilebilir. </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>

Çıkar çatışması ilan etti.

Lazer-aracılı yaralanma zebrafish embriyo kontrollü koşullarda rejenerasyon incelemek amacıyla hücreleri ablasyonu ile istenilen büyüklükte yaralar için güçlü bir yöntemdir. Özellikle, tam hücreler hedef olabilir (Şekil 2) ve yara alanı gibi zamanlama her ikisi de kontrol edilebilir. Daha sonra, yaralanma bölgesinde ve yenilenme işlemleri kolayca izlenir, kaydedilmiş (Şekil 3) ve (Şekil 1) analiz edilir. Lazer de x-ve y-ekseninde odaklanmıştır rağmen Ancak, tamamen z ekseni odaklı değildir. Bu ilgi hücreleri altında ya da üstünde yer alan hücrelere zarar verebilir. Bununla birlikte, bu yaklaşım kas tamir in vivo araştırmalarda geniş bir alanı açılır.

<table border="1"><tbody><tr><td> Isıtma bloğu </td><td></td><td></td><td></td></tr><tr><td> Çifti # 5 forseps </td><td> Dumont </td><td></td><td></td></tr><tr><td> Cam slayt </td><td> Menzel </td><td></td><td> 76 x 26 mm </td></tr><tr><td> Lameller </td><td> Roth </td><td></td><td> 50 x 24 mm # 1 </td></tr><tr><td> Vazelin </td><td></td><td></td><td></td></tr><tr><td> Stereomikroskopta </td><td> Leica </td><td></td><td> MZFLIII </td></tr><tr><td> Micropoint lazer </td><td> Andor Teknoloji </td><td></td><td></td></tr><tr><td> Flüoresans mikroskobu </td><td> Zeiss </td><td></td><td> Axioplan II </td></tr><tr><td> Metamorph yazılımı </td><td> Molecular Devices </td><td></td><td></td></tr><tr><td></td><td></td><td></td><td> Reaktifler <ul><li> Düşük erime noktalı agaroz (# 50081,Lonza) </li><li> Tricaine stok solüsyonu: 400 mg Tricaine (# A-5040, Sigma-Aldrich) / 100 ml dH 2 O pH 9.0 </li><li> E3, orta (5 mM NaCl, 0.17 mM KCI, 0.33 mM CaCl2, 0,33 mM MgSO4) </li></ul></td></tr></tbody></table>

laser-inflicted injury of zebrafish embryonic skeletal muscle

Çeşitli deneysel yaklaşımlar murin müsküler distrofi modelleri de myotoxin enjeksiyonları (bupivakain, cardiotoxin veya notexin), kas nakilleri (denervasyon-devaskülarizasyonun bağlı rejenerasyon), yoğun egzersiz, kas yenilenme çalışma amacı ile kas yaralanma ikna etmek için fare kullanılır, ama oylandı mdx fare (bu yaklaşımların bir inceleme için 1&#39;e bakınız) gibi. Zebrafish, genetik yaklaşımlar müsküler distrofi fenotipleri (örneğin runzel 2 ya da 3 sapje gibi) sergilerler ve distrofi ile ilişkili genlerin ekspresyonunu engellemek 4 morpholinos antisens oligonükleotid mutantları sayılabilir. Bunun yanı sıra, kimyasal yaklaşımlar ve böylece sonuçta müsküler distrofi 5 yol açar hypercontraction oluşur, bunların, aynı zamanda Galanthamine, bir asetilkolinesteraz inhibe edici kimyasal bileşik, örneğin mümkündür. Ancak, genetik ve farmakolojik yaklaşımlar gfiziksel yaralanmalar ölçüde daha kolay ve mekansal 1 kontrol edilir ise enerally, bireyin içindeki tüm kasları etkileyebilir. Lokalize fiziksel yaralanma bir iç kontrol olarak kontralateral kas değerlendirmesini sağlar. Başka bir grup son zamanlarda çok lokal bireyin embriyonik zebrafish kas plazma zarı zarar iki foton lazer (822 nm) kullanımı rapor Nitekim, biz son zamanlarda, zebrafish embriyo 6 iskelet kas yenilenme incelemek için lazer aracılı hücre ablasyon kullanılır hücreleri 7. Burada, zebrafish embriyo iskelet kas hücre hasarı için Micropoint lazer (Andor Teknolojisi) kullanımı için bir yöntem sunulmuştur. Micropoint lazer 435 nm dalga boyunda hedeflenen hücre ablasyon için uygun bir yüksek enerjili lazer. Lazer mikroskop aynı zamanda f da kullanılabilir ki bu tür bir şekilde (bizim kurulum, Zeiss bir optik mikroskop) bir mikroskop ile bağlanırveya numune üzerine ve yaralama (aydınlık veya floresans) etkileri görselleştirmek için lazer ışık odaklama. Lazer darbeleri kontrol parametreleri dalga boyu, yoğunluk ve bakliyat numarasını içerir. Şeffaflığını ve dış embriyonik gelişme nedeniyle, zebrafish embriyo lazer kaynaklı yaralanma ve hem de daha sonraki iyileşme incelemek için çok müsait olan. 1 ila 2 gün sonrası fertilizasyon, somitic iskelet kas hücreleri giderek kuyruk 8, 9 gövdesinden somitogenesis ilerlemesi nedeniyle posterior anterior olgunlaşması uğrarlar. Bu aşamalarda, embriyo kendiliğinden seğirme ve yüzme başlatabilir. Zebrafish yakın yetişkin zebrafish 10, 11 yaralanma sonrası rejenere edilebilir dokuların birçok türleri (vb vasküler, nöronal, kardiyak) gibi, doku rejenerasyon çalışması için önemli bir omurgalı model organizma olarak kabul edilmiştir.

Lazer-aracılı yaralanma immobilize 1 gün-yaşlı embriyolar üzerinde yapıldı. Şekil 1 &#39;de gösterildiği gibi, bir kaç lazer atımlarının normal olarak somite sınırları arasında gerilir hasarlı, sarmal, Aktin-zengin miyofibril tarafından kolayca tanınabilir, küçük bir yara oluşturabilir. Lazer atımlarının sayısı arttıkça, ancak en fazla miyofibril yok edilir bir ağır hasar somite blok neden olur. Yine de, biz kelimenin tam anlamıyla büyük olanlardan daha hızlı iyileşmesini küçük yaralanmalar ile, &quot;zaman bütün yaraları iyileştirir&quot; olduğunu gözlemleyebilirsiniz. İyileştikten sonra, Aktin renklendirmeler miyofibril tekrar normal somite yayılan olduğunu göstermektedir, ancak ek bilgi Xirp1 için boyama elde edilebilir. Xirp1 protein ektopik eskiden yaralandı kas hücreleri 6 içinde miyofibril iyileşmekte birlikte lokalize olarak yaralanma ve kurtarma, meydana gelmiş olduğu tespit için iyi bir göstergedir. Bazı amaçlar için, hücreler exactl belirlemek için önemlidiry lazer ile yaralandı, bu nedenle, genetik komşu hücrelere sahip olup olmadığının değerlendirilmesinde hassas olan hücre hedefli ve daha sonra görselleştirmek için edebilmek için bazı hücreler (mosaically bir GFP etiketli proteini ifade ederek örneğin) etiketlemek için yararlı olabilir lazer darbe (Şekil 2) ile de etkilenmektedir. Yara iyileşme ve antikor boyama sonra onu çevreleyen mosaically işaretli hücrelerin model sayesinde tekrar bulmak ve hedef hücre analiz etmek mümkün hale gelir. Bir ilginç yaklaşım, lazer aracılı yaralanma somitic kas nasıl etkilediğini zaman içinde düzenli olarak izlemektir. Bunun için, immobilize embriyo lazer yaralanma örneğin ilk dakika (Şekil 3) içinde hemen ortaya çıkan etkileri, görmek için canlı olarak kaydedilebilir. Bundan başka, kayıt timelapse hücrelerin yakından yeniden nasıl takip etmek için, arzu edilen herhangi bir zaman aralığı (örneğin 5 dakika aralığı) ile yapılabilmektedirYara yerinde. <img alt="Şekil 1" fo:content-width="5.5in" fo:src="/files/ftp_upload/4351/4351fig1highres.jpg" src="/files/ftp_upload/4351/4351fig1.jpg" /> Şekil 1. Verdirdiler yaralanma şiddeti göre, yaralanma kurtarma farkı adım aralıklarla devam edecektir. Uygulanan lazer darbeleri sayısına bağlı olarak büyük ölçüde değişebilir, ama sonunda, çoğu yara kapatılır. Biz son zamanlarda bu yaralanma üzerine upregulated ve 32 hpf 6 hasarlı miyofibril olmayan sarkomerik Aktin (kırmızı) lokalize olduğundan Xirp1 (yeşil), kas rejenerasyonu için iyi bir belirteç olduğunu göstermiştir. <img alt="Şekil 2," fo:content-width="6in" fo:src="/files/ftp_upload/4351/4351fig2highres.jpg" src="/files/ftp_upload/4351/4351fig2.jpg" /> Şekil 2. Etiketli hücreler özellikle (Ref 6 değiştirilerek) hedeflenebilir. Cells Tg mozaik ifade ile etiketlenmiş edildi [β-aktin: α-aktinin-GFP] (yeşil veya siyah beyaz). Böyle bir hücre 32 hpf burada hedef ve ciddi birkaç lazer darbeleri tarafından tahrip edilmiştir, gibi kırmızı cıvata ile gösterilir. Beyaz ok yaralı hücreyi gösterir; sarı oklar diğer GFP etiketli hücreleri gösterir. Sağdaki resim, aynı yara iyileşme ve antikor boyama sonra çekilmiştir. <img alt="Şekil 3" fo:content-width="2in" fo:src="/files/ftp_upload/4351/4351fig3highres.jpg" src="/files/ftp_upload/4351/4351fig3.jpg" /> Şekil 3,. Lazer yaralanma Canlı akışı edinimi yaralanma tüm somite blok nasıl etkilediğini ortaya koymaktadır. DIC ile canlı akışı modunda (yani yaklaşık 16 kare / sn) Axioplan kaydedilen tek resimlerin Montage. Lazer 28 hpf de somitic kas vurur yere siyah bir ok gösterir. Kırmızı oklar geri çekilmeden hasarlı uçları göstermektedirmyofibril. Biri somite blok içinde bu dinamik değişiklikleri 2,5 saniye içinde oluştu.

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

Burada sunulan yöntem, yüksek enerjili lazer darbeleri ve zamanla bu yaralanmalar ve kurtarma ileri analizi ile canlı zebrafish embriyo kesin hasar oluşur. Ayrıca genetik olarak tek etiketli veya iskelet kas hücre gruplarının sırasında ve lazer ışık kaynaklı hasar sonrası takip edilebilir göstermek.

Zebra balığı Embriyonik İskelet Kası Lazer-yaralama

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 Görüntüleme.  Max Delbrück Center for Molecular Medicine. Burada sunulan yöntem, yüksek enerjili lazer darbeleri ve zamanla bu yaralanmalar ve kurtarma ileri analizi ile canlı zebrafish embriyo kesin hasar oluşur. Ayrıca genetik olarak tek etiketli veya iskelet kas hücre gruplarının sırasında ve lazer ışık kaynaklı hasar sonrası takip edilebilir göstermek.

Video: 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.

Zebra balığı Embriyonik Göz Dokular, Mikrodisseksiyon

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 ...

Biyoloji

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.

Yetişkin ve Embriyonik İskelet, Kas Microexplant Kültür ve İskelet Kası Kök Hücre İzolasyonu

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.

İskelet Kası Mitokondrial İzolasyon

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.

İskelet Kası progenitörlerin saflaştırılması

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.

Zebra balığı Embriyonik Kalp Dissected, immün

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.

Disosiye Müstahzarlar gelen Embriyonik ve larval Zebra balığı İskelet myofibers Analizi

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.

Kalsiyum değerlendirilmesi Bozulmamış İskelet kas liflerinin içinde Sparks

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.

Zebra balığı Nephrotoxin Mikroenjeksiyon Akut Böbrek Yaralanması Model

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.

İskelet Kası Fibroblastlarından Eksozomların İzolasyonu ve Karakterizasyonu

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