Biz onun teknik yardım için Trent Waugh teşekkür etmek istiyorum. Biz de Hasta Çocuk ve bu proje için onların cömert finansmanı için Cure Konjenital Musküler Distrofi (CMD) için Hastanesi Pediatri Bölümü kabul etmiş sayılırsınız.

Agar Enjeksiyon Tabaklar hazırlanması 1. (Saat: 45 dk) <ol><li> E3 ortam içinde% 2 ila% 3 agaroz kaynatılır ve çözelti tezgah üzerinde hafifçe soğumaya bırakın. Not: hazırlanmakta olan enjeksiyon plakaların sayısı Gerekli agaroz miktarını belirler. Her bir enjeksiyon plakası agaroz solüsyonun yaklaşık 35 ml gerekmektedir. </li><li> Kaynatıldıktan sonra istenen sıcaklık enjeksiyon kalıp, üreticinin talimatlarına uygun olarak (örn., 45 ° C) elde edilene kadar agaroz soğumaya bırakın. </li><li> 100 mm&#39;lik bir çanak içine agaroz soğutuldu yaklaşık 35 ml dökün. </li><li> Çözeltisi içine Tercih enjeksiyon kalıp bir ucunu yerleştirin, ardından (bu hava kabarcıklarının oluşumunu azaltmaya yardımcı olacaktır) agaroz çözeltisi üzerine kalıp geri kalanı yatıyordu. </li><li> Agaroz çözelti, yaklaşık 30 dakika boyunca, ya oda sıcaklığında ya da 4 ° C katılaşmaya izin verin. </li><li> Katı agaroz kalıbın bir ucunu ayırmak için bir spatula kullanın. Yavaşça m kalanını kaldırmak<html> eski. </li></ol> Evans Blue Dye (EBD) Enjeksiyon Mix 2. Hazırlama (Zaman: 30 dk) <ol><li> 1X Ringer çözeltisi EBV bir% 1 stok olun (5 mM KCI, 155 mM NaCI, 2 mM CaCI2, 1 mM MgCI2, 2 mM Na 2 HPO 4, 10 mM HEPES, 10 mM glikoz, pH 7.2), bu, oda sıcaklığında saklanabilir. </li><li> 25 mg MW 10.000 kDa -dextran floresein izotiosiyanat bir stok çözeltisi (FITC) Marka / mL C ° -20 ° C&#39;de 1X Ringer çözeltisi ve mağaza </li><li> (. Örneğin, 100 ul&#39;lik bir son çalışma hacmi için: FITC dekstran stokunun 90 ul% 1 EMB&#39;nin 10 ul karıştırın) FITC-dekstran stokunun stok çözeltisi doğrudan% 0.1 EBD seyreltilerek püskürtme karışımı hazırlayın. </li><li> İyice vorteks enjeksiyon karışımı (yeşil açmalısınız) ve alüminyum folyo ile enjeksiyon karışımı tüp sararak doğrudan ışık tutmak. </li></ol> 3. EBD Enjeksiyon Hazırlama (Saat: Yaklaşık 30 dk) ent &quot;&gt; Not: Protokol 3-7 gün sonrası fertilizasyon (dpf) dan larvaları ile iyi çalışır. <ol><li> RT Ön sıcak enjeksiyon plakası. </li><li> Metal levha üzerine mikromanipülatör düzenleyerek enjeksiyon cihazı kurmak ve mikroskop sonraki enjeksiyon için kullanılan duruyor. Hava tahrikli mikroenjeksiyon kontrolörü açın. Not: Tercih edilen enjeksiyon sistemi laboratuvarı tarafından değişecektir ve analiz sonucunu değiştirmek gerekir. </li><li> Geri EBD karışımı yaklaşık 2-4 ul enjeksiyon iğnesi doldurun. </li><li> EBD karışımı yaklaşık 5 nl enjeksiyon hacmi kalibre. Not: Enjeksiyon hacmi kalibrasyonu kalibrasyon yöntemine bağlı olacaktır. Gaz basıncı enjektörler bir mikrometre kullanılarak hacimli yükleme dozu ile kalibre enjeksiyon hacmi gerekir, ancak bir pistonu tahrik enjeksiyonu ile doğrudan belirli bir enjeksiyon hacmi için ayarlanabilir. </li><li> 1X Ringer solüsyonu ile ıslak enjeksiyon plakası ve kuyulardan aşırı kaldırın. </li><li> % 0.04 etil 3-aminobenzoat metansülfonat sal ile önceden muamele larvalarıenjeksiyon başlamadan önce larva hareketsiz 1X Ringer çözeltisi içinde seyreltilmiş t (tricaine). Not: Uygun enjeksiyon herhangi bir kalıntı hareketi ile zor olduğu gibi larva sağlanması tamamen hareketsiz olan önemlidir. </li><li> Cam bir pipet kullanılarak agar enjeksiyon plakalarının oyuklarına anestezi larva yerleştirin. Larvalar tamamen kuyunun içinde ve onların yan yatarken olduğundan emin olun. Not: kuyu başına larva sayısı deneyci kadardır. </li><li> Larvalar kuyularda konur sonra, iyi olan larva hareketini en aza indirmek için aşırı Ringer çözüm kaldırmak. Bu larvalar dihidrat kalmamak çözelti bir artık miktarda bırakın. </li></ol> EBD ile Zebra balığı Larva 4. perikardiyal Enjeksiyon (Time: larvaları enjekte sayısına bağlı olarak, tahmin edilen 1-3 saat) <ol><li> Enjeksiyonlar yapılacak bir diseksiyon kapsamı larvaları içeren enjeksiyon plaka koyun. </li><li> Içeren enjeksiyon pipet iğne yerleştirinEBD bir Zebra balığı larvaları üzerinde karıştırın. </li><li> Yeniden pozisyonunda çevirerek enjeksiyon plakası yüzden enjeksiyon iğne larvaları kalbine yakın ve yaklaşık 45 ° ventral ön-arka eksen. </li><li> Damar başlangıçta dorsal yönde dönüyor sarısı (Şekil 1) ön kısmında ven bölgesinde ortak kardinal ven (CCV) içine enjeksiyon iğne takın. Not: en 40x arasında bir büyütme açık CCV görmek yararlı olabilir. </li><li> EBD karışımı 5 nl enjekte edilir ve EBD karışımı hemen sızıntısını en aza indirmek için 5-8 saniye pozisyonda iğne tutun. Not: İyi bir enjeksiyon kalp boşluklarına görülen boya renginin (Şekil 1) sahip olacaktır. EBV karışımı kalp gözlenmiştir değilse, o zaman EBV karışımı ilave bir 5 nl boya çekmesi uyarılması için yeterli olabilir enjekte. Alternatif olarak, embriyo atılabilir. Not: Bazı durumlarda, kalp dayak vermeyebilir. Bu o takdirdeccurs, 20-40 saniye larva izlemeye devam eder. Boya dolaşım sistemi içinde hareket Tipik olarak, kalp yenerek devam eder. </li><li> Bir sonraki larva ve tekrarı üzerine taşıyın. </li><li> Enjeksiyondan hemen (Şekil 2) sonra vaskülatürde FITC-dekstran varlığını gözlemleyerek başarıyla enjekte embriyolar belirleyin. </li></ol> 5. Kuluçka ve EBD Alımı (Saat: 4-6 saat) <ol><li> Larva istenilen sayıda enjekte edildikten sonra, geri 100 mm&#39;lik tabaklar içerisinde tricaine olmayan 1X Ringer çözeltisine larva enjekte edilmiştir. </li><li> Alüminyum folyo sarılı yemekler tutun. Not: Karanlıkta enjekte larvaların tutulması önemli ölçüde sağkalım oranlarını artırır ve sinyal gücü en büyük tutarlılık sağlar. Alüminyum folyo Sarma larva inkübatör dışındaki zaman süresi için özellikle önemlidir. </li><li> Yeterli EBD alımını sağlamak için larva 4-6 saat 28.5 ° C&#39;de inkübe izin verin. </li></ol&gt; Kas içinde EBD 6. Görselleştirme (Saat: larvaları enjekte ve mikroskopi Çeşidi sayısına bağlı olarak, tahmini 0,5-3 saat) <ol><li> Önce görüntüleme, hareket etmesini önlemek için% 0.04 tricaine ile larva uyutmak. </li><li> EBD alımı iskelet kası (Şekil 3) oluşup oluşmadığını kırmızı floresan altında Görünüm larvaları belirlemek için. </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>

Yazarlar hiçbir rakip mali çıkarlarını ya da diğer ilgi çatışmaları var.

Zebra balığı nöromüsküler hastalık 2,29 çalışma için güçlü bir araç olarak ortaya çıkmaktadır. Bugüne kadar, zebra balığı sistemi, yeni kas hastalığa neden olan mutasyonlar 16,17,30 doğrulamak yeni patolojik mekanizmalar 18 aydınlatmak ve potansiyel yeni tedavi edici ilaçlar 12,24 tanımlamak için kullanılmıştır. Bu kolektif çabalar, insan nöromüsküler hastalıkları modellemek için Zebra balığı yararını kurduk. Ancak, zebra balığı ve memeli modelleri ile yapılan ilerlemelere rağmen, nöromüsküler koşulları geniş spektrumu içinde hastalar için sınırlı tedavi seçenekleri vardır. Bu nedenle, yüksek bir talep yıkıcı hastalıkların bu grup için tedavi geliştirilmesine ihtiyaç vardır. Tedavi için bu talebi paralel yeni hayvan modelleri ve varsayılan tedavi stratejileri doğrulamak için sürekli deneysel yenilik yanı sıra titiz analiz için gelen bir ihtiyaçtır. EBD analizi yaygın fare modellerinde kullanılanÇalışma dokusu ve beyin, kalp hücresel hasar ve iskelet kası 27,31. En önemlisi, EBD kas zarı istikrarsızlık şiddetini göstermek ve 8 zarar çeşitli kas distrofi alt tiplerinin fare modellerinde yaygın olarak kullanılır. Kas zarı hasarı ortaya çıkarmak için EBD kullanımı, insan hastalık durumuna 9 hayvan modelinin benzerlik kuran destekleyici bir parametredir. Fare EBD güç geliştirmek ve nöromüsküler hastalık zebrabalıkları modellere EBD uygulamak, kendi dahil olmak üzere birçok laboratuarları, yol açmıştır. Nedeniyle EBD analizinin uygulanabilirliği, bu tekniğin etkin insan hastalık durumuna 11,15,22,24,32 için Zebra balığı modellerini doğrulayacak uygulanmaktadır. Hasarlı kas zarlarla Larva kas lifleri içinde EBD alımı ve dolayısıyla kırmızı floresan sahip olacaktır. Tek kas lifleri içindeki ara-lif alan gözlenen floresans değil, aynı zamanda, t bazal membran ayırma liflerin bilgi verici olabiliryararlı tanı detay sağlayan membran hasar o yokluğu. EBD analizi hayvan modeli doğrulama ötesinde potansiyel uygulama vardır. Bizim laboratuarından çabalar son zamanlarda EBD analizi potansiyel yeni tedavi ilaçları 24 doğrulayarak yararlı olduğunu göstermiştir. Potansiyel tedavi tedaviler azaltmak veya ilgili terapötik eylem 8 delalet edebilir nöromusküler hastalık modellerinde EBD alımını kaldırılması durumunda belirleme. Bu tip analiz terapötik mekanizması (ler) kurmaya yardımcı ve EBD analizi uygulaması genişletir olabilir. Birçok teknikleri ile olduğu gibi, EBD analizi deneysel tasarım ve uygulama sırasında dikkat edilmesi gereken birkaç uyarılar var. Örneğin, nedeniyle yaş doku kalınlaşması ccv belirlemek zor olabilir. Buna ek olarak, bu deney sayısını azaltmak ve larvaların sayıda hazırlık için ihtiyaç artmaktadır önce ve perikardiyal enjeksiyon sırasında hazırlanmasında larvalarına kolaydır.Hasarlı kas EBD sürebilir Dahası, taşıma ve enjeksiyon sırasında larva yapılan fiziksel hasar yanlış pozitif sonuç verebilir. Bu engellerin bazılarının üstesinden gelmek için, biz enjeksiyon ve önceki sonraki analiz için aşağıdaki derhal başarılı boya infüzyonu ile larva kolay ve güvenilir şekilde belirlenmesini sağlar, bu video makalede bir ko-enjeksiyon stratejisi nitelendirdiler. Önce kas lifleri tarafından alımına vaskülatürde EBD onayını vererek başarılı enjeksiyon için ko-enjeksiyon kontrolleri FITC-dekstran. EBV floresans kas lifleri toplanan takdirde birkaç saat sonra larva oldukça yaygın hale gelir, bu durum özellikle faydalı olabilir; gibi, bu tespit etmek zor olabilir. Buna ek olarak, CCV eksik ve yumurta sarısı veya vücut boşluğu içine enjekte EBD, inkübasyondan sonra, hasarlı kas lifleri tarafından alım olasılığının azaltılması ile, henüz kontrol embriyolarına benzer bir difüz-kırmızı flüoresans neden olabilir. Topluca, bu mağaraats EBD enjeksiyon, tutarlı ve güvenilir sonuçlar elde etmek için sabır ve pratik gerektirir öneririz. Toplamda, biz Zebra balığı larvaları üzerinde EBD analizi gerçekleştirmek için pratik ve basit bir yöntem açıklanmaktadır. Bugüne kadar, bir model sistem olarak zebrabalıkları kullanımı, özellikle de bir insan hastalığı modeli olarak, hızlı bir şekilde genişlemektedir. Bu genişleme zebrabalıkları sisteminin mevcut avantajları üzerine geliştirmek deneysel tekniklerin sürekli gelişme ve değişiklik kısmen kaynaklanmaktadır. EBD enjeksiyon tekniği doğrulama ve Zebra balığı kas hastalığı modellerinin çalışma için bir araştırmacının cephanelik ek ve güçlü bir araç sağlar. Bu tekniğin devam eden uygulama ve modifikasyon yeni tedavi stratejilerinin yanı sıra hastalığa neden olan mekanizmaları açığa çıkarmalarına yardımcı potansiyeline sahiptir.

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.

EBV püskürtme karışımı 3 dpf sapje homozigoz mutantları ve vahşi tip kardeş CCV enjekte edilmiştir. Kalp odacıkları (Şekil 1B) doldurulmuş Enjeksiyonlar sonra FITC-dekstran yeşil floresan altında damarsal (Şekil 2) görselleştirmek başarılı enjeksiyon için analiz edildi. 4 saat enkübasyon süresinden sonra, EBV alım floresan mikroskopi kullanılarak hücre gruplarının düzeyde incelenmiştir. Sapje ​​homozigoz mutantlar, kas zar 15 (Şekil 3B) zarar gösteren, EBV alımı gösterdi, oysa, vahşi tipli kardeşlerine, görünür bir kas lifleri (Şekil 3A) içinde herhangi bir EBV floresans sergilenmektedir. <img alt="figür 1" src="/files/ftp_upload/53183/53183fig1.jpg" /> Bir Zebra balığı Embry ortak kardinal ven (CCV) içine Şekil 1. Damariçi EBD enjeksiyon karışımıo. (A) uninjected embriyo. Ok CCV enjeksiyon için ideal bir konuma işaret eder. (B) CCV içine Başarılı enjeksiyon. Boya kalp boşlukları (ok) girer ve damar pompalanması başlar. (C) Bir başarısız CCV enjeksiyonu bazı veya embriyo (ok) sarısı kesesi giren boya tüm sonuçlanacaktır. <a href="https://www.jove.com/files/ftp_upload/53183/53183fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/53183/53183fig2.jpg" /> Şekil 2. Embriyolar enjeksiyondan hemen öncesinde EBD alımı için aşağıdaki damarsal boyunca yeşil floresan altında FITC-dekstran dağıtım gözlemleyerek başarılı enjeksiyon için düzenlenmiş olabilir.Bu rakamın büyük halini görmek için lütfen buraya tıklayınız. <img alt="Şekil 3," src="/files/ftp_upload/53183/53183fig3.jpg" /> Şekil 3: REF hasarlı membran ile elyaflar tarafından ele alınacaktır kas liflerinin herhangi bir EBV floresan gösteren (A) Vahşi tipli kardeşlerine.. (B) Birden fazla kas lifleri (oklar) içinde EBD floresan ile Sapje ​​homozigot mutant. Bütün larvalar REF püskürtme karışımı ile enjekte edilmiş ve 3 dpf bir 4 saatlik bir bekletme sürecinden sonra analiz edilmiştir. Kardeşleri ve mutantlar CCV enjeksiyondan önce kas lifi dekolmanı göre sınıflandırılmaktadır bulundu. <a href="https://www.jove.com/files/ftp_upload/53183/53183fig3large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</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.

Evans Blue Dye ile Zebra balığı Larva İskelet Kası Bütünlüğü Analizi

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 Görüntüleme.  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.

Time-lapse aydınlık stereomikroskobi ile Zebra balığı Larva Doku Onarım yakalama

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 ...

Gelişim Biyolojisi

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.

Cardiotoxin Enjeksiyonu ile Akut İskelet Kası Regeneration indüksiyon

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.

Microstructured cihazlar için en iyi duruma getirilmiş mikroenjeksiyon ve Zebra balığı larva görüntüleme

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.

Contractile fare faaliyet kaynaklı kas iskelet fenotipik uyarlamalar çalışmaya kronik stimulasyon uygulanması

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.

Kas iskelet uydu hücreleri tarafından Pax7 ve Laminin antikorlar ile ayirt tanımlaması

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.

İskelet Kas Biyopsisi Prosedüründen İnsan Kas Atası Hücrelerinin İzolasyon, Kültür, Karakterizasyonu ve Farklılaşması

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.

İmmün eksik farelerde İnsan İskelet Kası Ksenogreftlerinin

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.

Fare İskelet Kas Ekstranında Primer Miyoblastların İzolasyon ve Farklılaşması

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.

Zebrabalığı Embriyoları ve Larvalarında Tüm Mount Immunohistochemistry

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.

Kas Hastalığının Zebra Balığı Modellerinde Kas Yenilenmesinin İncelenmesi

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