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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Methods. 6, 875-881 (2009).</li><li>Zhou, W., 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=Non-sense+mutations+in+the+dihydropyridine+receptor+beta1+gene,+CACNB1,+paralyze+zebrafish+relaxed+mutants.">Non-sense mutations in the dihydropyridine receptor beta1 gene, CACNB1, paralyze zebrafish relaxed mutants.</a> Cell Calcium. 39, 227-236 (2006).</li><li>Dora, K. A., Doyle, M. P., Duling, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elevation+of+intracellular+calcium+in+smooth+muscle+causes+endothelial+cell+generation+of+NO+in+arterioles.">Elevation of intracellular calcium in smooth muscle causes endothelial cell generation of NO in arterioles.</a> Proc. Natl. Acad. Sci. 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 border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		</tr>
		<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>CO2 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 &#956;m Filter</td>
			<td>BD Biosciences</td>
			<td>352340</td>
			<td></td>
		</tr>
		<tr>
			<td>70 &#956;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-&#945;-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

Research

JoVE Journal

Biology

11.5K 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

Primary Dissociated Midbrain Dopamine Cell Cultures from Rodent Neonates

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation from systemic input from elsewhere in the brain. In our lab, we use these neurons to assess dopamine release kinetics using carbon fiber amperometry, as well as expression levels of dopamine related genes and proteins using quantitative PCR and immunocytochemistry. In this video, we show you how we generate these cultures from rodent neonates.
The process involves several steps, including the plating of cortical glial astrocytes, the conditioning of neuronal cell culture media by the glial substrate, the dissection of the midbrain in neonates, the digestion, extraction and plating of dopamine neurons and the addition of neurotrophic factors to ensure cell survival.
The applications suitable for such a preparation include electrophysiology, immunocytochemistry, quantitative PCR, video microscopy (i.e., of real-time vesicular fusion with the plasma membrane), cell viability assays and other toxicological screens.

Primary dissociated midbrain dopamine cell cultures allow for the study of presynaptic characteristics of dopamine neurons. They can be used to monitor real-time dopamine release kinetics and protein/mRNA levels of regulators of dopamine exocytosis. Here, we show you how to generate these cultures from rodent neonates.

小学游离中脑多巴胺鼠类新生儿的细胞培养

The ability to create primary cell cultures of dopamine neurons allows for the study of the presynaptic characteristics of dopamine neurons in isolation ...

科研

生物学

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

从胚胎斑马鱼和cDNA合成的基因表达分析的RNA分离

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

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.

斑马鱼胚胎眼组织显微切割的

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 ...

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.

成人和胚胎骨骼的肌肉Microexplant文化和骨骼肌肉干细胞的分离

Cultured embryonic and adult skeletal muscle cells have a number of different uses. The micro-dissected explants technique described in this chapter is a ...

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

幼虫，少年和成年斑马鱼的心脏解剖，斑马鱼</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

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.

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 ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Isolation and Culture of Individual Myofibers and their Satellite Cells from Adult Skeletal Muscle

Muscle regeneration in the adult is performed by resident stem cells called satellite cells. Satellite cells are defined by their position between the basal lamina and the sarcolemma of each myofiber. Current knowledge of their behavior heavily relies on the use of the single myofiber isolation protocol. In 1985, Bischoff described a protocol to isolate single live fibers from the Flexor Digitorum Brevis (FDB) of adult rats with the goal to create an in vitro system in which the physical association between the myofiber and its stem cells is preserved 1. In 1995, Rosenblattmodified the Bischoff protocol such that myofibers are singly picked and handled separately after collagenase digestion instead of being isolated by gravity sedimentation 2, 3. The Rosenblatt or Bischoff protocol has since been adapted to different muscles, age or conditions 3-6. The single myofiber isolation technique is an indispensable tool due its unique advantages. First, in the single myofiber protocol, satellite cells are maintained beneath the basal lamina. This is a unique feature of the protocol as other techniques such as Fluorescence Activated Cell Sorting require chemical and mechanical tissue dissociation 7. Although the myofiber culture system cannot substitute for in vivo studies, it does offer an excellent platform to address relevant biological properties of muscle stem cells. Single myofibers can be cultured in standard plating conditions or in floating conditions. Satellite cells on floating myofibers are subjected to virtually no other influence than the myofiber environment. Substrate stiffness and coating have been shown to influence satellite cells' ability to regenerate muscles 8, 9 so being able to control each of these factors independently allows discrimination between niche-dependent and -independent responses. Different concentrations of serum have also been shown to have an effect on the transition from quiescence to activation. To preserve the quiescence state of its associated satellite cells, fibers should be kept in low serum medium 1-3. This is particularly useful when studying genes involved in the quiescence state. In serum rich medium, satellite cells quickly activate, proliferate, migrate and differentiate, thus mimicking the in vivo regenerative process 1-3. The system can be used to perform a variety of assays such as the testing of chemical inhibitors; ectopic expression of genes by virus delivery; oligonucleotide based gene knock-down or live imaging. This video article describes the protocol currently used in our laboratory to isolate single myofibers from the Extensor Digitorum Longus (EDL) muscle of adult mice (6-8 weeks old).

Isolation and culture of myofibers is the gold standard in vitro system to study the transition of satellite cells through quiescence, activation and differentiation. Importantly, the single myofiber culture system preserves the myofiber/stem cell association, which is an essential component of the muscle stem cell niche.

单个肌纤维和成人骨骼肌卫星细胞的分离和培养

Muscle  regeneration in the adult is performed by resident stem cells called satellite  cells. Satellite cells are defined by  their position between the ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

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.