作者要感谢道林实验室的成员（亚伦Reifler，特伦特沃，安吉拉布斯塔，和威廉特尔弗）​​促成了技术的发展和生产的手稿。这项工作是由该研究所陶布曼，小儿科的部门在密歇根大学，并在部分从肌肉萎缩症协会（JJD MDA186999）和美国国立卫生研究院（JJD 1K08AR054835）资助。

1。聚-L-赖氨酸的制备涂层盖玻片（时间:1小时） 涂层盖玻片允许快速肌纤维的沉降和附着。这可能在肌纤维隔离（下面的步骤2）的解离步骤中进行。 <ol><li>切割和封口膜放置在60毫米培养皿（任何品牌）的底部。 </li><li>放置显微镜盖玻璃卡瓦（12毫米直径）上的Parafilm在60mm组织培养皿或单独放置在单个井的24孔板中。 </li></ol>注1:这些小圆形盖玻片有助于集中肌纤维数量和减少多余抗体的使用。 注2:其他大小的盖玻片也行，不过，使用的试剂和胚胎的体积将需要进行相应的调整。 <ol start="3"><li>移液器50-200微升的聚-L-赖氨酸上的每个玻璃盖在培养皿溶液（0.01％）。 </li><li>让盖玻片坐至少一个浩乌尔在37°C再孵育不会产生负面影响的结果。 </li><li>从玻璃盖除去聚-L-赖氨酸溶液中，用最少的100μl的1×PBS中洗两次。 </li><li>允许盖玻片干燥。 </li><li>盖玻片现在已经准备好为肌纤维。 </li></ol>注3:为了确保无菌，涂层可以在发动机罩和高压灭菌的盖玻片进行。 注4:保持底部60毫米培养皿用封口膜。这种设置将肌纤维电镀和免疫标记（后面的步骤）期间使用。 选择1:相反涂层盖玻片在使用前立即的，涂覆的盖玻片库存都可以使用。可以存储在包含众多盖玻片4℃含有至少2毫升聚L-赖氨酸60毫米培养皿。用这种替代从步骤1.5处理盖玻片肌纤维。 方案2:我们也聚-L-鸟氨酸大衣盖玻片。 Ŧ他是劳动密集型的，而且是用于较长期培养有用的，因为多聚-L-鸟氨酸涂布的盖玻片上可以是紫外线处理。用紫外线治疗和精心无菌技术活肌纤维通常可以维持在培养4-7天。 2。斑马鱼胚胎和电镀肌纤维（1至3小时的时间）的解离注:标准协议适用最好3旦（天受精后）胚胎。 <ol><li>转移斑马鱼胚胎成一个标准的1.5 ml离心管中，去除尽可能多的多余的鱼水之成为可能。通常情况下，每管10-20胚，虽然少都可以使用。使用超过20-25胚胎通常导致过度的制备密度。 <ol><li>删除之前解离绒毛膜，手动使用＃5镊子。另外，化学绒毛膜去除与一个10-15分钟链霉蛋白酶处理来实现。通常情况下，以前的去除绒毛膜只需要当MUT事先确认蚂蚁的表型或形态是必需的，或者使用阶段时，其中胚胎是自然地从他们的绒毛膜孵化。 </li></ol></li><li>加入900微升的CO 2的独立媒体，以含有胚胎的试管。 </li><li>加入100μl胶原酶II型，终浓度3.125毫克/毫升，开始解离（31.25毫克/毫升稀释在1×PBS中免胶原酶溶液）。 Ⅳ型胶原酶也可用于分离。 </li><li>旋转在轨道摇床胚胎，并使用P1000的Pipetman的研磨磨碎，每30分钟，在室温。 </li></ol>注2:仔细监测胚胎分离;过高或过低解离（尤其是以上）是协议失败的最常见原因。次消化将取决于研磨，每管胚胎数，和胚胎的年龄的强度而变化。它也往往较少（相比于野生型），用于从骨骼肌突变体胚胎。 注3:每胚龄平均消化时间:1 DPF = 1小时，2 DPF = 1.5小时，3 DPF = 2小时，4 DPF = 2-2.5小时。 <ol start="5"><li>终止消化时，没有整个胚胎是可见的，但坚固件仍清晰可见- 这是必不可少的，以防止过 ​​量。 </li><li>离心管中，以离解的胚胎在0.8〜xg离心3-5分钟以沉淀细胞。 </li><li>从沉淀的细胞除去上清液，用CO 2独立媒体洗2倍。 </li><li>加入新鲜的CO 2独立媒体重悬细胞。 1毫升通常用于10-20胚胎的棉片。的体积可以根据胚胎数进行缩放。 </li><li>使用P1000的移液管，通过一个70微米的过滤器通过胚胎停牌。这有助于从预习中删除不需要的杂物。 </li></ol>注4:胚胎悬浮液进行过滤，第二次通过一个40微米的过滤器，以进一步去除不必要的杂物。从双重过滤，回收是逼近的ately 800微升从1毫升的起始体积。 <ol start="10"><li>加约50-100微升肌纤维悬浮液的各聚-L-赖氨酸包被的盖玻片。 </li></ol>注5:用封口膜底部执行在培养皿中的步骤2.9，先前准备的。该封口膜保持运行脱涂盖玻片的肌纤维悬浮液。保持覆盖，以防止水分蒸发的培养皿中。 <ol start="11"><li>允许肌纤维沉降约1小时到涂覆的盖玻片在室温下。 </li></ol>注6:肌纤维将开始在5-10分钟解决。然而，良好的肌纤维附着，最低1小时（1-2小时）的建议。使肌纤维定居更长不会伤害准备。对于较长的孵育（包括过夜），抗生素可以被添加到该介质。用抗生素和无菌技术的活培养物通常可以维持1-2天。 <ol start="12"><li>现场米yofibers可以在这一点上被观察到。从胚胎2旦肌纤维和以后将被看作是横纹肌和伸长的细胞（ 图1）。此时，肌纤维现在已经准备好现场分析或免疫标记。 </li></ol>注7:从1骨骼肌DPF胚胎不板作为拉长，并明确横纹肌纤维。相反，大myoballs是可见的。此外，丸粒化阶段后的解离过程中（步骤2.6），1旦myoballs要被离心的最低的8分钟，在5000×g离心来实现沉淀。胚胎在此阶段的分析，它是推荐使用表达EGFP特异性骨骼肌23的转基因株系。这将允许鉴定细胞的肌肉起源与其他来源。 3。游离斑马鱼肌纤维的荧光免疫染色（时间:约1天） 3.1。免疫标记<ol><li>除去的部分从肌纤维粘附到涂覆的盖玻片媒体</li><li>用4％多聚甲醛或甲醇固定细胞。对于PFA，取出半的媒体音量，更换有PFA的量相同。修正了在室温下10分钟，然后取出总量，取代50-100微升的PFA，并编定一个额外的10分钟。对于酒精固定，冰冷甲醇或丙酮可以在4℃，分别施加10分钟或5分钟。 </li><li>洗肌纤维至少3到5倍用1×PBS或1x PBS加0.1％吐温。一般洗涤量为每盖玻片50-100微升。 </li><li>加入封闭液，以肌纤维（股份合作），并孵育20-60分钟，在室温下。封闭液将根据第一和第二抗体而异。两种最常见的在实验室中使用的是（1）的1×PBS，2毫克/毫升牛血清白蛋白，1％绵羊血清，0.25％的Triton X-100的最终和（2）0.2％的Triton X-100，2毫克/毫升（0.2 ％BSA）和5％绵羊/山羊血清。 </li><li>除去封闭液，加入​​稀释为B一抗锁定的解决方案或PBS。过夜孵育肌纤维在4℃下请务必要么封口膜或包裹在保鲜膜含有涂有抗体的肌纤维装盖玻片的培养皿中。可以添加小件浸湿纸巾来创建一个加湿室。 </li><li>从盖玻片除去第一抗体和洗涤盖玻片3 - 5×5分钟，用PBS或阻断溶液。 </li><li>加入二抗，适当的浓度稀释在1×PBS或1-2小时在室温封闭液。 </li><li>去除二级抗体并在PBS中，每次5分钟，在室温洗肌纤维3〜5倍。 </li></ol> 3.2。安装盖玻片<ol start="9"><li>适用于1-2滴抗淬灭剂对载玻片。 </li><li>使用镊子和地点（肌纤维向下）盖玻片上的载玻片的抗淬灭剂仔细回升的涂层和immunolabled盖玻片。 </li></ol>注1:注意到c的方向oated盖玻片是至关重要的。 <ol start="11"><li>躺在1-2的Kimwipes在坚硬的固体表面。快速反转与涂盖玻片搁在抗淬灭剂的幻灯片，并将其放置（盖玻片下）上的Kimwipes。 </li><li>轻轻按压在显微镜幻灯片的角落，以除去多余的抗淬灭，并形成滑动和盖玻片之间的密封</li><li>允许残留干燥在室温5-10分钟的抗淬灭，然后将肌纤维准备图像（ 图2A和B），或储存于4℃。 </li></ol> 4。活细胞钙成像使用GCaMP3 活细胞的实验可以在肌纤维前固定（以下步骤2.10）来进行。以下协议描述了肌纤维表达GCaMP3 24，一种基因编码的钙指示剂，由骨骼肌特异性的斑马鱼α-肌动蛋白启动子（pSKM）23表达的实时成像。另外，这种技术可以很容易地适合于使用钙指示剂染料如Fura-2的。 <ol><li>注射胚胎pSKM:GCaMP3构建在1-2细胞期</li><li>在3 DPF，收集幼虫和在第1和2中所述准备肌纤维。使肌纤维附着了至少1小时的。对于该技术中，在24孔培养皿中制备盖玻片是必需的。 </li><li>小心地取出多余的介质，并加入300微升新鲜的CO 2的独立媒体在室温。 </li><li>观察在倒置显微镜下的细胞。 GCaMP3阳性肌纤维应根据绿色荧光可见。 </li><li>设置相机用于记录，如果需要的话。 </li><li>准备中的CO 2独立媒体30毫米咖啡因的解决方案。为刺激细胞，轻轻吸取300微升的咖啡因溶液倒入井中下放大倍率。在5-10秒，GCaMP阳性肌纤维应该响应与咖啡因（ 图3），在荧光的快速增长。 MyofibERS也可能收缩。值得注意的是，咖啡因诱导的肌浆网卖场25钙释放。其它试剂，如氯化钾26和兰尼4，可用于研究诱导的钙释放。 </li></ol>

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U.S.A. 94, 6529-6534 (1997).</li><li>Hirata, H., 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=accordion,+a+zebrafish+behavioral+mutant,+has+a+muscle+relaxation+defect+due+to+a+mutation+in+the+ATPase+Ca2++pump+SERCA1.">accordion, a zebrafish behavioral mutant, has a muscle relaxation defect due to a mutation in the ATPase Ca2+ pump SERCA1.</a> Development. 131, 5457-5468 (2004).</li><li>van Eeden, F. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mutations+affecting+somite+formation+and+patterning+in+the+zebrafish,+Danio+rerio.">Mutations affecting somite formation and patterning in the zebrafish, Danio rerio.</a> Development. 123, 153-164 (1996).</li><li>Gupta, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+dag1+mutant:+a+novel+genetic+model+for+dystroglycanopathies.">The zebrafish dag1 mutant: a novel genetic model for dystroglycanopathies.</a> Hum. Mol. Genet. 20, 1712-1725 (2011).</li><li>Leuranguer, V., Papadopoulos, S., Beam, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organization+of+calcium+channel+beta1a+subunits+in+triad+junctions+in+skeletal+muscle.">Organization of calcium channel beta1a subunits in triad junctions in skeletal muscle.</a> J. Biol. Chem. 281, 3521-3527 (2006).</li><li>Capote, J., Bolanos, P., Schuhmeier, R. P., Melzer, W., Caputo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calcium+transients+in+developing+mouse+skeletal+muscle+fibres.">Calcium transients in developing mouse skeletal muscle fibres.</a> J. Physiol. 564, 451-464 (2005).</li><li>Adams, B. A., Tanabe, T., Mikami, A., Numa, S., Beam, K. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intramembrane+charge+movement+restored+in+dysgenic+skeletal+muscle+by+injection+of+dihydropyridine+receptor+cDNAs.">Intramembrane charge movement restored in dysgenic skeletal muscle by injection of dihydropyridine receptor cDNAs.</a> Nature. 346, (1990).</li><li>Sakowski, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+approach+to+study+motor+neurons+from+zebrafish+embryos+and+larvae+in+culture.">A novel approach to study motor neurons from zebrafish embryos and larvae in culture.</a> J. Neurosci. Methods. 205, 277-282 (2012).</li><li>Nakano, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis+of+GPI-anchored+proteins+is+essential+for+surface+expression+of+sodium+channels+in+zebrafish+Rohon-Beard+neurons+to+respond+to+mechanosensory+stimulation.">Biogenesis of GPI-anchored proteins is essential for surface expression of sodium channels in zebrafish Rohon-Beard neurons to respond to mechanosensory stimulation.</a> Development. 137, 1689-1698 (2010).</li><li>Davidson, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+deletion+of+lysine+7+expands+the+clinical,+histopathological+and+genetic+spectrum+of+TPM2-related+myopathies.">Novel deletion of lysine 7 expands the clinical, histopathological and genetic spectrum of TPM2-related myopathies.</a> Brain. 136, 508-521 (2013).</li><li>Schredelseker, J., Shrivastav, M., Dayal, A., Grabner, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-Ca2+-conducting+Ca2++channels+in+fish+skeletal+muscle+excitation-contraction+coupling.">Non-Ca2+-conducting Ca2+ channels in fish skeletal muscle excitation-contraction coupling.</a> Proc. Natl. Acad. Sci. U.S.A. 107, 5658-5663 (2010).</li></ol>

作者宣称没有利益冲突。

斑马鱼是研究体内 25,27,28肌肉发育和功能的有力脊椎动物模型系统。他们也成为一笔宝贵的财富用于模拟人类肌肉疾病14,15,20,29。虽然大踏步已采取提前斑马鱼的肌肉功能和肌肉疾病的研究的使用和应用，有一个恒定的迫切需要开发工具，让更多的深入分析，赞美的遗传，形态，行为和功能优点斑马鱼已经提供17。因此，我们已经适应，并开发了一个简单和强大的技...

骨骼肌是负责生成所必需的蠕动收缩力高度专业化的组织。收缩是通过称为兴奋-收缩（欧共体）耦合，转换电信号从细胞内贮存1,2钙释放的过程开始。细胞内钙释放激活的肌节缩短和产生力量。负责调解神经肌肉接头处传递3，EC耦合​​4,5，和肌动蛋白-肌球蛋白依赖性收缩6的分子机器的许多具体的组件继续深入研究的持续主题。此外，在收缩7,8和介导的肌纤维和细胞外基质7,9之间的信令稳定肌细胞膜蛋白已经确定，并研究了很详细。 在许多基因的肌肉结构的重要突变ND功能已被确定为人体骨骼肌肉疾病的原因（ <a href="http://www.musclegenetable.org/" target="_blank">http://www.musclegenetable.org/</a> ）。这些疾病，大致分类为基础的临床和病理特征骨骼肌肌病和肌营养不良症，都与肌肉无力，终身残疾，早期死亡率10,11相关联。斑马鱼已被证明是用于建模和研究人类骨骼肌肉疾病8,12,13一个优秀的系统。它已被用来验证新的基因突变8，确定疾病的病理生理学14,15的新内容，并找出新的治疗方法15,16。斑马鱼研究人类肌肉疾病的功率涉及大量的后代，肌肉结构和功能，斑马鱼胚胎的光学透明度，并且易于显影斑纹的遗传药理学和操纵的快速发展的鱼17。 我们和其他人12,18,19最近开发出一种简单的技术为肌纤维来自发展中国家斑马鱼的快速，高效的隔离。这种方法使肌纤维的考试比可以通过全胚胎分析提供更多细节。该技术已被开发用于蛋白质亚细胞定位20以及表征为重要病理特征为在新开发的疾病模型21的验证研究的一部分的识别。此外，孤立的肌纤维还可以用于实时成像和电生理研究22，技术，允许对肌肉功能的关键方面的审讯。的特定协议为肌纤维隔离，随着后续的分析实验的两个例子，在该原稿的其余部分详细说明。

<table><tbody><tr><td>24-well culture plate</td><td>Corning</td><td>3524</td><td></td></tr><tr><td>10x PBS</td><td>Invitrogen Gibco</td><td>70011</td><td></td></tr><tr><td>CO&lt;sub&gt;2&lt;/sub&gt; Independent medium</td><td>Invitrogen Gibco</td><td>18045</td><td></td></tr><tr><td>Collagenase Type II</td><td>Worthington Biochemical</td><td>LS004186</td><td>Lot 41H12764</td></tr><tr><td>Collagenase Type IV</td><td>Worthington Biochemical</td><td>L5004188</td><td></td></tr><tr><td>8% Paraformaldehyde</td><td>Electron Microscopy Sciences</td><td>157-8</td><td></td></tr><tr><td>Methanol</td><td>Sigma</td><td>322415</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>X100</td><td></td></tr><tr><td>BSA</td><td>Sigma</td><td>A2153</td><td></td></tr><tr><td>Sheep serum</td><td>Sigma</td><td>S3772</td><td></td></tr><tr><td>Goat serum</td><td>Sigma</td><td>G9023</td><td></td></tr><tr><td>Glass coverslips</td><td>Fischerbrand</td><td>12-545-82 12CIR-1D</td><td></td></tr><tr><td>Poly-L-Lysine</td><td>Sigma</td><td>P4707</td><td></td></tr><tr><td>Pronase</td><td>Sigma</td><td>P5147</td><td></td></tr><tr><td>40 &amp;mu;m Filter</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>70 &amp;mu;m Filter</td><td>BD Biosciences</td><td>352350</td><td></td></tr><tr><td>Prolong Gold antifade reagent</td><td>Invitrogen</td><td>P36931</td><td></td></tr><tr><td>Anti-&amp;alpha;-Actinin antibody</td><td>Sigma</td><td>A5044</td><td></td></tr><tr><td>Anti-RYR antibody</td><td>Abcam</td><td>34C</td><td></td></tr><tr><td>Alexa Fluor antibody</td><td>Invitrogen</td><td>A-21425</td><td></td></tr><tr><td>TWEEN 20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>60 mm Petri dish</td><td>Fischerbrand</td><td>0875713A</td><td></td></tr><tr><td>Poly-L-Ornithine</td><td>Sigma</td><td>P4957</td><td></td></tr><tr><td>Microscope slide</td><td>Fischerbrand</td><td>12-550-15</td><td></td></tr><tr><td>Caffeine</td><td>Sigma</td><td>C0750</td><td></td></tr></tbody></table>

analysis of embryonic and larval zebrafish skeletal myofibers from dissociated preparations

斑马鱼已被证明是一个有价值的模型系统研究骨骼肌功能和研究人类肌肉疾病。尽管通过体内分析骨骼肌在斑马鱼提供的，可视化的复杂和精细结构的蛋白质环境负责的肌肉功能，尤其是在整个胚胎中的诸多优点，可能会产生问题。这个障碍源于斑马鱼的骨骼肌（60微米）和肌节的甚至更小尺寸的小尺寸。在这里，我们描述和演示从斑马鱼胚胎和幼虫分离骨骼肌纤维的简单和快速的方法。我们还包括了协议，说明后期制备技术用于分析的肌肉结构和功能非常有用。具体来说，骨骼肌蛋白质我们详细介绍了随后的免疫细胞化学定位，并通过活细胞钙成像刺激钙释放的定性分析。总体来说，这款视频本文提供了斑马鱼的骨骼肌纤维的分离和表征，一种技术，它提供了一种导管，用于肌肉结构和功能的无数的后续研究的直向前和有效的方法。

肌纤维的荧光免疫染色（ 图2） 从显示预期的肌纤维荧光标记图形图像成功分离和电镀后免疫染色。在肌纤维已被标记为与抗兰尼碱受体（1:100）（ 图2A）或抗α-辅肌动蛋白（1:100）（ 图2B）的抗体，并分别揭示三联及Z带的免疫染色。用二抗的Alexa Fluor 555（1:500）。图像利用共聚焦显微镜拍摄的。 从肌纤维...

Watch this Scientific Journal Video about Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations at JoVE.com

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

斑马鱼是用于模拟骨骼肌的人类疾病的一个新兴的系统。我们描述了一个快速，高效的方法从胚胎和幼虫的斑马鱼隔离骨骼肌肌纤维。这种方法产生了高密度的肌纤维制备适合的单骨骼肌纤维形态的研究中，蛋白质的亚细胞定位，以及肌肉生理学。

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

analysis-embryonic-larval-zebrafish-skeletal-myofibers-from

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.6K 次观看.  University of Michigan. 斑马鱼是用于模拟骨骼肌的人类疾病的一个新兴的系统。我们描述了一个快速，高效的方法从胚胎和幼虫的斑马鱼隔离骨骼肌肌纤维。这种方法产生了高密度的肌纤维制备适合的单骨骼肌纤维形态的研究中，蛋白质的亚细胞定位，以及肌肉生理学。 

视频: Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

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.

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.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

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

科研

发育生物学

Using Touch-evoked Response and Locomotion Assays to Assess Muscle Performance and Function in Zebrafish

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many myopathies affecting skeletal muscle function can be quickly and easily assessed in zebrafish over the first few days of embryogenesis. By 24 hr post-fertilization (hpf), wildtype zebrafish spontaneously contract their tail muscles and by 48 hpf, zebrafish exhibit controlled swimming behaviors. Reduction in the frequency of, or other alterations in, these movements may indicate a skeletal muscle dysfunction. To analyze swimming behavior and assess muscle performance in early zebrafish development, we utilize both touch-evoked escape response and locomotion assays.
Touch-evoked escape response assays can be used to assess muscle performance during short burst movements resulting from contraction of fast-twitch muscle fibers. In response to an external stimulus, which in this case is a tap on the head, wildtype zebrafish at 2 days post-fertilization (dpf) typically exhibit a powerful burst swim, accompanied by sharp turns. Our method quantifies skeletal muscle function by measuring the maximum acceleration during a burst swimming motion, the acceleration being directly proportional to the force produced by muscle contraction.
In contrast, locomotion assays during early zebrafish larval development are used to assess muscle performance during sustained periods of muscle activity. Using a tracking system to monitor swimming behavior, we obtain an automated calculation of the frequency of activity and distance in 6-day old zebrafish, reflective of their skeletal muscle function. Measurements of swimming performance are valuable for phenotypic assessment of disease models and high-throughput screening of mutations or chemical treatments affecting skeletal muscle function.

Zebrafish are an excellent model to study muscle function and disease. During early embryogenesis zebrafish begin regular muscle contractions producing rhythmic swimming behavior, which is altered when the muscle is disrupted. Here we describe a touch-evoked response and locomotion assay to examine swimming performance as a measure of muscle function.

采用触摸诱发反应和运动测定以评估在斑马鱼肌肉性能和功能

Zebrafish muscle development is highly conserved with mammalian systems making them an excellent model to study muscle function and disease. Many ...

Building Finite Element Models to Investigate Zebrafish Jaw Biomechanics

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical strain. Skeletal joints are subjected to dynamic mechanical loading. Finite element analysis (FEA) is a computational method, frequently used in engineering that can predict how a material or structure will respond to mechanical input. By dividing a whole system (in this case the zebrafish jaw skeleton) into a mesh of smaller 'finite elements', FEA can be used to calculate the mechanical response of the structure to external loads. The results can be visualized in many ways including as a 'heat map' showing the position of maximum and minimum principal strains (a positive principal strain indicates tension while a negative indicates compression. The maximum and minimum refer the largest and smallest strain). These can be used to identify which regions of the jaw and therefore which cells are likely to be under particularly high tensional or compressional loads during jaw movement and can therefore be used to identify relationships between mechanical strain and cell behavior. This protocol describes the steps to generate Finite Element models from confocal image data on the musculoskeletal system, using the zebrafish lower jaw as a practical example. The protocol leads the reader through a series of steps: 1) staining of the musculoskeletal components, 2) imaging the musculoskeletal components, 3) building a 3 dimensional (3D) surface, 4) generating a mesh of Finite Elements, 5) solving the FEA and finally 6) validating the results by comparison to real displacements seen in movements of the fish jaw.

Finite Element Analysis is a frequently used tool to investigate the mechanical performance of structures under load. Here we apply its use to modeling the biomechanics of the zebrafish jaw.

建立有限元模型，研究斑马鱼下颌生物力学

Skeletal morphogenesis occurs through tightly regulated cell behaviors during development; many cell types alter their behavior in response to mechanical ...

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.

检查斑马鱼肌肉疾病模型中的肌肉再生

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

Real Time and Repeated Measurement of Skeletal Muscle Growth in Individual Live Zebrafish Subjected to Altered Electrical Activity

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish with fluorescently-marked plasma membranes can be scanned in a confocal laser scanning microscope in order to determine the volume of muscle tissue and the number of muscle fibers present. Efficient approaches for the measurement of cell number and size in live animals over time are described and validated against more arduous segmentation methods. Methods are described that permit the control of muscle electrical, and thus contractile, activity. Loss of skeletal muscle contractile activity greatly reduced muscle growth. In larvae, a protocol is described that allows reintroduction of patterned electrical-evoked contractile activity. The described methods minimize the effect of inter-individual variability and will permit analysis of the effect of electrical, genetic, drug, or environmental stimuli on a variety of cellular and physiological growth parameters in the context of the living organism. Long-term follow-up of the measured effects of a defined early-life intervention on individuals can subsequently be performed.

Optical clarity is a major advantage for cell biological and physiological work in zebrafish. Robust methods for measurement of cell growth in individual animals are described that permit novel insights into how growth of skeletal muscle and neighboring tissues are integrated with whole body growth.

实时和重复测量电活动改变的个体活斑马鱼的骨骼肌生长

A number of methods can be used to visualize individual cells throughout the body of live embryonic, larval or juvenile zebrafish. We show that live fish ...

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.

Immunostaining of Dissected Zebrafish Embryonic Heart

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.

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

生物学

Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of myosin heavy chain (MyHC) isoforms has emerged as a finer discriminator of fiber-type. Type I, type IIA, type IIX and type IIB fibers can now be identified with precision based on their MyHC profile; however, manual analysis of these data can be slow and down-right tedious. In this regard, rapid, accurate assessment of fiber-type composition and morphology is a very desirable tool. Here, we present a protocol for state-of-the-art immunohistochemical staining of MyHCs in frozen sections obtained from mouse hindlimb muscle in concert with a novel semi-automated algorithm that accelerates analysis of fiber-type and fiber morphology. As expected, the soleus muscle displayed staining for type I and type IIA fibers, but not for type IIX or type IIB fibers. On the other hand, the tibialis anterior muscle was composed predominantly of type IIX and type IIB fibers, a small fraction of type IIA fibers and little or no type I fibers. Several image transformations were used to generate probability maps for the purpose of measuring different aspects of fiber morphology (i.e., cross-sectional area (CSA), maximal and minimal Feret diameter). The values obtained for these parameters were then compared with manually-obtained values. No significant differences were observed between either mode of analysis with regards to CSA, maximal or minimal Feret diameter (all p &#62; 0.05), indicating the accuracy of our method. Thus, our immunostaining analysis protocol may be applied to the investigation of effects on muscle composition in many models of aging and myopathy.

Immunohistochemical staining of myosin heavy chain isoforms has emerged as the state-of-the-art discriminator of skeletal muscle fiber-type (i.e., type I, type IIA, type IIX, type IIB). Here, we present a staining protocol along with a novel semi-automated algorithm that facilitates rapid assessment of fiber-type and fiber morphology.

小鼠骨骼肌形态及纤维型成分的半自动化分析

For years, distinctions between skeletal muscle fiber types were best visualized by myosin-ATPase staining. More recently, immunohistochemical staining of ...

医学

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

在收缩力测量评估肌肉功能在斑马鱼幼虫

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

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 ...

Isolation of Cardiac and Vascular Smooth Muscle Cells from Adult, Juvenile, Larval and Embryonic Zebrafish for Electrophysiological Studies

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the zebrafish cardiovascular tissues have been limiting in studying their electrophysiological properties. Previous methods have been described for dissection of zebrafish hearts and isolation of ventricular cardiac myocytes. However, the isolation of zebrafish atrial and vascular myocytes for electrophysiological characterization was not detailed. This work describes new and modified enzymatic protocols that routinely provide isolated juvenile and adult zebrafish ventricular and atrial cardiomyocytes, as well as vascular smooth muscle (VSM) cells from the bulbous arteriosus, suitable for patch-clamp experiments. There has been no literary evidence of electrophysiological studies on zebrafish cardiovascular tissues isolated at embryonic and larval stages of development. Partial dissociation techniques that allow patch-clamp experiments on individual cells from larval and embryonic hearts are demonstrated.

The present protocol describes the acute isolation of viable cardiac and vascular smooth muscle cells from adult, juvenile, larval, and embryonic zebrafish (Danio rerio), suitable for electrophysiological studies.

从成年、幼年、幼虫和胚胎斑马鱼中分离心脏和血管平滑肌细胞以进行电生理研究

Zebrafish have long been used as a model vertebrate organism in cardiovascular research. The technical difficulties of isolating individual cells from the ...