This publication is funded through Cure CMD, an Association Contre Les Myopathies (AFM) grant (project 16297), and the National Institutes of Health (grant numbers K08 AR059750 and L40 AR057721). We would also like to thank Dr. Julie Tetzlaff for her assistance in revising this manuscript.

标记被用于肌肉存储为相应的标识符对动物和肌肉收获的容器。预冷却袋/容器和工具，以防止组织冻结/解冻它被添加时。 1，研究设计注意事项肌肉组织收集<ol><li>对于小动物肌肉疾病（鼠）模型，使用整个肌肉的研究，因为小动物模型往往需要一个完整的肌肉进行评估，以进行病理研究提供足够的肌纤维取样。对于大型动物（犬）模型的肌肉疾病，使用肌部是在其横向（横截面）的高宽至少0.3×0.3 cm以上。 注:核心活检也可以使用，但可能是次优由于采样问题的主要表征的研究。 </li><li>对于涉及肌节的研究中，肌节长度的超微结构测量结果可包括临界端点，并且可以热曲愤怒专门处理。这是通常只在相关的疾病如肌节演出长度不合适的元素，如杆状体肌病的一些原因。一些实验室偏好来修复肌肉在其非收缩或伸展状态，而不是在非张紧或者非拉伸肌肉执行这些测量。 <ol><li>如果肌小节长度将无法测量，修复肌肉组织没有直接将它放到所需的EM固定液（见试剂）预拉伸肌肉。 </li><li>如果肌节的长度将被测量，可以使用多种技术来预拉伸的肌肉（见讨论）: <ol><li>使用一个夹紧装置，以保持肌肉在其静止张力而它仍然是在动物，切除肌肉围绕夹的外侧，并直接将其放置到所需的EM固定液（见试剂）。 </li><li>伸展和脚的肌肉的表面（如一块软木）在类似于所观察到的长度<eM&gt;体内后，已提取的动物。 </li></ol></li></ol></li></ol> 2，子划分隔离骨骼肌成片段是适合所需的研究（图1） <ol><li>对于冰冻肌肉组织学: <ol><li>包括尽可能多的给定的肌肉尽可能地减少抽样偏差（见步骤1.1），但标本应&lt;1.​​5厘米避免冻结文物。 </li><li>对待所有的肌肉从同一研究同样以防止纤维尺寸伪迹的差异是由于处理分歧。 </li></ol></li><li>用于电子显微镜（EM）: <ol><li>切〜0.5×0.2×0.2cm的肌肉从样品条带，用平行于原始肌肉的纵向轴线的样品的纵向轴线。 注:固定剂将不能充分穿透较厚的组织片，因此保持试样的厚度为2mm或更小是在获得适当的结果是至关重要的。此外，如肌肉的这种小片段的定向是非常困难的，它最好是使用组织的模仿肌肉的实际取向（ 即，固定的试样的最长方面是肌肉/肌纤维的纵向方向），以一个片段最大限度地减少电磁处理过程中处理和混乱。 </li></ol></li><li>细胞培养: <ol><li>所需的细胞产量可能影响肌肉的所需细胞培养建立的量。单一的肌肉（肌肉或部分）由小动物可提供足够的细胞数量，建立细胞培养，所以如果需要的话，从多个池中的肌肉组织。 </li></ol></li></ol> 3，组织固定和处理电子显微镜（EM） <ol><li>放置肌肉不大于2毫米的最薄维度直接到期望的EM固定液更大（参见试剂）的片段。 </li><li>将在新兴市场固定缓冲液的肌肉2-48小时fixati后（见试剂）上。长期固定（几个星期或几个月）会导致超微结构细节上的电磁研究的损失。 </li><li>发送到EM核心设施进行处理。 </li></ol> 4，肌凝的组织学，生物化学和分子生物学研究<ol><li>通过彻底印迹检体用纸巾除去过多的水分在试样直至组织稍微粘附在毛巾。过多的水分会在肌肉产生显著冻结文物。 注:组织可以通过滚动它在婴儿爽身粉被进一步干燥。此步骤一般是不需要的肌肉，除非它们已经在过度潮湿的环境中。 </li><li>放置十月（或类似的粘合剂，如西黄蓍胶）中的浅cryomold的底部或软木，只有足够十月以便为导向的肌肉（ 图2C）的基础。标签冻结前的软木或cryomold可能是标本跟踪有用。 </li><li> Caref来自ully放置肌入模具中所需的方向，与大多数肌肉从十月突出，使得剖开肌肉不与十月（ 图2C）相接触。 </li><li>添加异戊烷到金属杯，直到达到约3-4厘米的深度。 </li><li>戴上热防护手套，取出杜瓦的盖子。 </li><li>放置或保持在金属杯与液氮接触，从而使液氮在杯的外侧上的电平高于异戊烷在杯子内部的水平。策略来安排这些容器，如图2所示。 <ol><li>不要让液氮进入金属杯，因为它产生的气泡的泡沫，可以用繁琐的工作（也是不够寒冷冻结）。 </li></ol></li><li>观察异戊烷，以确定何时已经达到适当的温度。在适当的温度（最佳的-140至-149℃），使冷冻异戊烷盖白色卵石将形成（几分钟后，并在最初的雾已经异戊烷以上消失）在杯子的底部，或在溶液中一般会变厚，以糖蜜的一致性。冻结之前，这个阶段将产生结冰atifacts。 <ol><li>如果异戊烷完全冻结坚实，解冻，并将其寒意后续使用前再次冻结温度。 </li></ol></li><li>用预冷的镊子，降低标本和软木/模具放入异戊烷约10-20秒。 </li><li>将冷冻的样品到预先冷却的容器中。 </li><li>保持试样和容器上的干冰在任何时候，直到转移到-80℃冰箱。 </li></ol> 5，如果冷冻神器是目前在已经冷冻的组织，它可以提高冷冻神器的等级使用下面的"解冻和重新冻结"程序<ol><li>取块出来的冷冻机， 其中一个在一个时间，并让他们在干冰。本协议的时间是至关重要的。只解冻和重新冻结一个块的时间。 </li><li>移动样品从干冰至室温。 </li><li>如果样品是嵌入在华侨城，用手术刀为彻底清除周围华侨城越好。 </li><li>允许样品在室温下解冻到它是完全解冻的点，而不是在那里变干的地步。 <ol><li>轻轻地督促试样用钳子，以确保试样进展到下一个步骤之前完全解冻。 </li></ol></li><li>冻结肌肉如步骤4.1-4.10规定。 </li></ol> 6制备肌肉的剥皮单纤维功能测试<ol><li>对肌肉的存储过程取决于当肌肉/纤维被用于实验上。 <ol><li>对于在几个星期内使用时，将肌肉中的50％glycerol/50％放松溶液中的溶液（见试剂），并储存在-20℃。 </li><li>使用尾部呃几个星期，将肌肉中的75％glycerol/25％放宽溶液的溶液（见试剂），并储存在-80°C。 </li><li>或者，让肌肉干，脚软木，和存储在二氧化硅颗粒在-80°C。这些脱水肌肉可以使用多年。 </li></ol></li></ol> 7，准备新鲜的肌肉的细胞培养一批货物到实验室外协议为建立细胞培养已在别处4-7良好描述。 <ol><li>填在50ml的无菌锥形管中，用无菌的完全生长培养基。 </li><li>用无菌镊子，完全浸没在介质中的组织加肌肉组织的管。 <ol><li>填写与媒体管的剩余部分，​​以防止运输过程中的干燥。 </li></ol></li><li>加冰袋的保丽龙盒，然后将锥形管附近几个冰袋。 <ol><li>请注意，只有冰袋（而不是干冰）应使用以防止损坏从液体和组织的冰冻。 </li></ol></li><li>寄送包裹的O / N，但组织可以持续在这些条件下2天。 </li></ol>

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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=Mutations+of+tropomyosin+3+(TPM3)+are+common+and+associated+with+type+1+myofiber+hypotrophy+in+congenital+fiber+type+disproportion.">Mutations of tropomyosin 3 (TPM3) are common and associated with type 1 myofiber hypotrophy in congenital fiber type disproportion.</a> Hum Mutat. 31, 176-183 (2010).</li></ol>

作者什么都没有透露。

骨骼肌是一个结构和功能上独特的组织，并专门编写的程序是必要的，以允许的结构和功能参数的最佳评估。而各种组织中通常冻结在临床和研究上下文病理研究，对于非肌肉组织冷冻协议冷冻前通常涉及组织在OCT中的全浸没。如示于图3中 ，这样的协议不适合于骨骼肌的病理评价，但足以类似于这里所描述的协议，这是一个经常遇到的错误。本文的目标是提供一个简单的协议，用于肌肉的妥善处理，以避免这样的问题。建议也被编上肌肉异地生理和细胞研究的妥善处理，努力促进收购的高质量的数据在案件以外的核心实验室磨片重新现场研究是不可取的或可能的。 作为在该协议指出，这是在肌肉的正确处理绝对必不可少的病理研究内容包括最小化的组织中的水含量，从而降低在该肌肉被冻结的温度，并增加在该冷冻达到的速度。组织或冷冻过程（由温度不足或缺乏的冷冻剂和所述组织之间的直接接触，产生如遇到用液氮）的过度缓慢内过多的水分会导致冻结的工件，可以削弱病理分析。华侨城提供了组织液的额外来源，许多实验室使用其他粘合剂像西黄蓍胶作为嵌入基板。即使在被适当地执行冻结的情况下，应注意通过与RT容器或器具接触，以避免意外后解冻标本。因而，一个成功的冻结​​过程需要一定程度的规划，其中包括一个预冷所使用的所有仪器和容器。当遇到冷冻的工件，有这里描述的是足以满足大多数病理学评估的组织的恢复方法。然而，这种冷冻/解冻循环不提供完美的组织学和有损害的组织（除了需要重新冻结的组织中的时间）的其它分子或酶研究的电位，因此，使用适当的初始冷冻做法是大大优选。在冷冻工件被预先冷冻的组织中遇到的情况下，然而，这里描述的技术可以是非常有用的。 固定组织的EM可以提供，此前组织收集需要规划具体的技术挑战和处理。采集标本的EM时最常见的错误包括使用组织碎片太厚的glutaraldehyd的e来渗透。戊二醛只从一个给定的表面，护理穿透约0.1厘米进入肌肉组织，应采取措施，确保了EM的样本中的一维是厚度不超过0.2厘米。此外，由于EM是直接评估收缩装置的极好手段，一些研究者已经开发战略，以预拉伸或预拉伸前的肌肉固定，使收缩元素的测量在生理相关的张力。没有标准协议，用于预张紧，但两种策略在本协议简要介绍。应当指出，试图预张紧的肌肉会产生不可预测的结果，除非它们被做在一个非常特定的方式，并且它可能是优选的修复肌肉松驰状态，以防止在肌小节长度伪迹的变化通过一个非标准预张紧步骤8,9。对于肌肉在这种特定的测量是没有必要的（包括第二个白用于临床目的进行iopsies），努力以预张力的肌肉一般不进行，并且对肌肉形态的主要效果是肌节的肌非均匀间距。 本文代表了第一个系列提供标准操作程序进行测试的先天性肌肉疾病领域的表现，它代表了20位专家在执行例行的细胞，分子，功能性，生理性的先天性肌肉疾病领域的努力，病理研究。发表的SOP的范围内将提供在未来的一年，并为每个必要的协议和相应的出版形式，这在先天性肌肉疾病协会研讨会于2013年四月在华盛顿举行讨论这个SOP努力的目标是提供路线图进行必要的测试和样本分析中的先天性肌肉疾病领域1）标准化在我们的领域作为米的做法和终点UCH越好，2）提供指导标准的做法，新的研究人员在我们的领域。我们相信，这些资源将促进新的调查进入我们的下深入研究的领域，从而提高了可进行的研究范围之内。此外，实践标准化将是比较不同的研究数据，并在规划和实施临床前和临床试验时，识别端点极有帮助。 而本文的重点是关系到适当的冻结和准备组织的各种研究，我们的合作小组还讨论了有用的端点的肌肉标本的病理分析。目前，正在执行的病理分析时采用的方法没有官方的共识，以及各种不同的研究分析，以与新的发现之前的出版物为每个相应的疾病。因此，我们认为这将是有益的建议在肌肉病理特征病理端点的规划一些一般性的准则。前量化的一项研究的病理学，相当大的思想应放入1）纤维尺寸测量的方法，2）的纤维类型特定异常或治疗效果的可能性，3）异常或影响的可能性被限制个别的肌肉，和4）的策略用于定量病理结果是特征的疾病正在研究中。纤维的大小是一个必要的端点大多数研究，遗憾的是在它是如何量化广泛的变化。许多研究利用专有的图像处理软件提供的自动定量方法报告这些结果，但许多这些项目的拍摄快捷键（如假设纤维是圆形或椭圆形），可以使这些自动测量不准确。有必要了解这些自动化的程序是如何让他们的测量结果b安伏有信心的测量，我们鼓励研究者，包括纸张的方法这些细节。此外，用于表示纤维大小的特定的测量是非常可变的，有些测量是优选他人10,11。纤维的大小的常用测量纤维横截面面积（CSA），尤其是因为进行生理学研究调查其结果标准化，以使用其工具获得CSA测量。不幸的是，虽然CSA测量可以准确地反映纤维尺寸完美横截面，它们广泛地依赖于纤维方向（的范围内，纵向或斜向剖面的纤维将有虚高CSA测量）和对纤维的大小从而不理想的测量。纤维尺寸的优选测量其较少依赖于纤维的截面积为最小的Feret直径（MinFeret直径），这是个测量e小调直径在肌肉细胞12。这种测量方法是仅稍微依赖于纤维的取向和一般临床黄金标准纤维的测量，调查被鼓励移向使用这种技术。这些测量通常可以使用其生成CSA测量值13中的相同的软件制造，并且也简单的手动测量。对于根据光纤类型，特定的肌肉，并在涉及到具体的疾病病理结果的情况下评估的病理资料，这些都是应该只是一个研究的规划过程中考虑较少争议的问题。纤维类型可以用免疫组织化学或ATP酶染色法进行评价，但要考虑到特定的肌肉和动物物种具有这些纤维类型的特定混合物（因而需要不同的期望和测试策略）是有用的。肌肉特异性病理介入或治疗功效可以发生，并且总肌重量与对照相比，可以用来决定肌肉病理评估之前，以确定疾病的异质性程度。最后，这是众所周知的，许多肌肉 ​​疾病都与特定的病理异常（如在杆状体肌病杆状体杆）14,15相关联，因此它也是有用的，考虑这些异常是否被发现在纤维型或肌在进行分析时，16,17的具体分布。总体而言，虽然我们不建议一个不灵活的一套肌肉的评价标准，但我们相信这些问题之前，应在任何骨骼肌肉疾病的病理研究的性能考虑。

Skeletal muscle is a unique tissue because of its structure and function, which can present a number of challenges when evaluating its pathology. While it is sufficient, and often even optimal, to evaluate most tissues following formalin fixation and paraffin embedding, this process leads to the production of cell shrinkage artifacts that impair the evaluation of critical phenotypes in congenital muscle disease (including myofiber size and shape). Additionally, frozen muscle is required when performing many histochemical stains (including the reduced nicotinamide adenine dinucleotide (NADH), cytochrome oxidase (COX), succinate dehyrogenase (SDH), adenosine triphosphatase (ATPase), and Oil Red O stains) that are essential for the detection of specific pathological abnormalities required to diagnose a variety of congenital myopathies, mitochondrial myopathies, and storage diseases. Many antibodies and molecular tools used to characterize muscle morphology and function have consequently been developed and optimized only for frozen tissue, which further limits the usefulness of fixed tissue preparations in studies of skeletal muscle biology. As a result, it has become standard practice to freeze skeletal muscle specimens for most clinical and research indications in the field of congenital muscle disease, despite the technical challenges that appropriate freezing might pose.
The freezing of skeletal muscle can be technically challenging due to the high likelihood of ice crystal formation within myofibers when an inappropriate freezing protocol is followed1. In most cases, freezing artifacts are encountered when the water content of the tissue is too high or when the freezing process is too slow. The water content of tissue can be accidentally increased by immersion in fluid, such as transport of the muscle in saline; or freezing can be compromised if the tissue has been entirely immersed in OCT. Both of these procedures can lead to catastrophic freezing artifacts in muscle tissue. Additionally, a number of small missteps in the freezing process may cause slow freezing or accidental thawing of samples after appropriate freezing, and these issues can similarly complicate pathological evaluation. 
As new models and treatments for skeletal muscle disease are developed, it will become increasingly useful to use standard operating procedures (SOPs) to evaluate skeletal muscle phenotypes and treatment efficacy. This is particularly important for the pathological evaluation of congenital muscle diseases, as the recognition of subtle phenotypes seen in many of these disorders can be impaired by improper tissue preparation. This report represents an effort by a Congenital Muscle Disease Consortium to establish a skeletal muscle freezing SOP for the study of congenital muscle disease models. 
The protocol presented here describes a strategy used in our clinical and research labs to optimize assessment of frozen muscle pathology, while also being suitable for a variety of protein-based and genetic studies. Specific strategies to prepare skeletal muscle specimens for electron microscopy are also presented. Additionally, as single fiber functional testing of frozen tissue is possible2,3, experts in these techniques have provided details on optimal tissue handling protocols. Unfortunately, the isolation of myoblast cell lines cannot be effectively performed on pre-frozen tissue, so a strategy is presented herein for the short-term storage and transport of muscle tissue for myoblast culture establishment at outside laboratories. Overall, the methods described here provide guidelines for the appropriate processing of muscle tissue for a variety of pathological, functional, molecular and cellular studies, regardless of the on-site capabilities of a given laboratory.

<table border="0" cellpadding="0" cellspacing="0" width="1111">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">For tissue freezing</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td style="width:369px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:523px;">Liquid nitrogen Dewar with a liquid withdrawal device</td>
			<td style="width:107px;">Custom Biogenic Systems</td>
			<td style="width:115px;">Lab-5 Series</td>
			<td>Any other liquid nitrogen dewar could substitute.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Cold-conductive container</td>
			<td style="width:107px;">Fisher</td>
			<td style="width:115px;">02-583A</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Wide insulated container capable of holding liquid nitrogen</td>
			<td style="width:107px;">Nalgene</td>
			<td style="width:115px;">4150-2000</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:523px;">Oakton Temp 10 Thermocouple Thermometer</td>
			<td style="width:107px;">Thomas Scientific</td>
			<td style="width:115px;">1228Y01</td>
			<td>Optional, but can be very useful in identifying the point at which isopentane is freezing.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">For single fiber functional testing</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:523px;">Petri dish</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:115px;">08-757-11</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:523px;">Sylgard coating</td>
			<td style="width:107px;">Ellsworth Adhesives</td>
			<td style="width:115px;">&#160;3-6636</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:523px;">Dissection stereomicroscope</td>
			<td style="width:107px;">Harvard Apparatus</td>
			<td style="width:115px;">693101</td>
			<td>Any other dissection microscope could substitute.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">For cell culture</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:523px;">Sterile laminar flow hood</td>
			<td style="width:107px;">Thermo Scientific</td>
			<td style="width:115px;">51022485</td>
			<td>Any other cell culture hood could substitute.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Materials</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">For tissue freezing</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Plastic bags/containers</td>
			<td style="width:107px;">Nasco</td>
			<td style="width:115px;">B01009WA</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Thermal safety gloves</td>
			<td style="width:107px;">Denville</td>
			<td style="width:115px;">G4162</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:523px;">Face shield</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:115px;">S47640</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:523px;">Long forceps</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:115px;">&#160;12-460-807</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Marker</td>
			<td style="width:107px;">Staples</td>
			<td style="width:115px;">125328</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">For cell culture</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Sterile 50 mL conical tubes</td>
			<td style="width:107px;">Denville</td>
			<td style="width:115px;">C1060-P</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">PES filter unit, 500 mL, 0.22 &#181;m</td>
			<td style="width:107px;">Denville</td>
			<td style="width:115px;">F5227</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:523px;">Sterile forceps</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:115px;">644320</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:523px;">Ice packs</td>
			<td style="width:107px;">Fisher Scientific</td>
			<td style="width:115px;">NC9909223</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Insulated Styrofoam box</td>
			<td style="width:107px;">Polar Tech</td>
			<td style="width:115px;">207F</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Packing tape</td>
			<td style="width:107px;">Staples</td>
			<td align="right" style="width:115px;">795570</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:523px;">Reagents</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Tissue Freezing</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:523px;">Liquid nitrogen</td>
			<td style="width:107px;">Airgas</td>
			<td style="width:115px;">NI NF160LT350</td>
			<td>Store liquid nitrogen in containers designed for low-temperature liquids</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Isopentane (2-methylbutane)</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">M32631-4L</td>
			<td>Store Isopentane&#160; in a flammable materials cabinet.&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:523px;">EM fixative (choose one)</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td style="width:369px;">The buffers without fixative should also be available, as EM-fixed tissues should be transferred from fixative to buffer after several hours or days if they are not immediately processed for EM.</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:523px;">2.5% glutaraldehyde in 0.1M cacodylate buffer</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:115px;">16537-15</td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:523px;">Karnovsky&#39;s fixative (3% glutaraldehyde + 2% paraformaldehyde in 0.1M phosphate buffer)</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:115px;">15732-10</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">For functional studies (if desired)&#160;</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Relaxing solution, containing (in mmol/L)</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 40 BES</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">B9879</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 10 EGTA</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">E4378</td>
			<td></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;">&#160;&#160;&#160;&#160;&#160; 6.56 MgCl2</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">M8266</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 5.88 Na-ATP</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">A3377</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 1.0 DTT</td>
			<td style="width:107px;">RPI</td>
			<td style="width:115px;">D11000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 46.35 K-propionate</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td>Make a solution by mixing proprionic acid (Sigma P1386) and KOH (Fisher P250) 1:1</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 15 creatine phosphate, pH 7.0</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">P7936</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 0.4 leupeptin</td>
			<td style="width:107px;">Calbiochem</td>
			<td style="width:115px;">10895</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 0.1 PMSF</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">P7676</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Skinning solution, containing&#160;</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; Relaxing solution (See Above)</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 0.5% Triton X-100</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">T8787</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Indicating silica granules</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Flat pieces of cork (1 x 1 inch)</td>
			<td style="width:107px;">Electron Microscopy Sciences</td>
			<td style="width:115px;">63305</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Glycerol</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">G2025</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">For cell culture (if desired)</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Complete Growth Medium, containing (for 500 mL)</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td>Sterilize by filtering the solution through a 500 mL 0.22 &#181;m PES filter unit.&#160; Store at 4&#176;C and use within 1 month.</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 395 mL of high glucose Dulbecco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">D5671</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 100 mL of fetal bovine serum (FBS)&#160;</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">F6178</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">&#160;&#160;&#160;&#160;&#160; 5 mL of 100X Penicillin-Streptomycin-Glutamine (PSG)</td>
			<td style="width:107px;">Sigma</td>
			<td style="width:115px;">G1146</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;"></td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:523px;">Storage requriements</td>
			<td style="width:107px;"></td>
			<td style="width:115px;"></td>
			<td></td>
		</tr>
		<tr height="20">
			<td colspan="3" height="20" style="height:20px;">See the materials safety data sheet (MSDS) for all other reagents for storage specifications</td>
			<td></td>
		</tr>
	</tbody>
</table>

tissue triage and freezing for models of skeletal muscle disease

骨骼肌是因为它的结构和功能，这需要用于组织收集特定的协议来从功能性的，细胞，分子和病理评估获得最佳结果的唯一组织。由于见于先天性肌肉疾病以及潜在的供固定干预承认这些特征的一些病理异常的微妙，当评估骨骼肌先天性肌肉疾病冷冻肌肉病理评价，优选固定的肌肉。此外，为了产生严重的冰冻文物肌肉的潜在需要特定的预防措施，冻结骨骼肌冻结其他组织时并不常用组织学检查时。此手稿描述用液氮冷却以保持最佳的骨骼肌形态的协议为使用异戊烷（2 - 甲基丁烷）骨骼肌的快速冷冻。这个过程也有效于Freezing组织用于基因或蛋白表达的研究。此外，我们还整合了冷冻协议到一个更广泛的过程，还介绍了组织的短期分流首选方法（1）单根纤维的功能性研究及（2）成肌细胞培养，重点是收集必要的最低限度的努力组织并运输到专门的研究或参考实验室来完成这些研究。总体而言，这份手稿提供了如何新鲜组织可以有效地分布于各种表型研究的概况，从而为相关的先天性肌肉疾病的病理研究提供了标准操作规程（SOP）。

为了提供可见不当冻结文物的例子，几个鼠标股四头肌采用不同的技术（ 图3）复制的一般常见的错误冻结。 如图3A所示 ，适当的冷冻骨骼肌显示肌纤维的紧密并置到周围组织（通常是其它的肌纤维，但有时肌纤维之间的肌内膜空间填充有纤维变性或炎性细胞在多种疾病或损伤过程中）和一个清晰可见细胞质车厢。对于病理分析，以查看细胞内空间的能力往往比肌内膜车厢更为重要，因为它清楚地识别的退行性变化，再生变化，或其他特殊病征的发现（中央细胞核，细胞内的夹杂物）在此的存在通常是至关重要的空间。因此，冰晶在细胞内隔室的存在表示点在肌肉的病理学评估ajor问题。 适当冷冻肌肉组织的同时需要足够冷的温度和冷冻，以避免冰晶形成足够的速度。占这两个因素，即使，相当大的冷冻神器仍然可以发生，因为过多的水分内的肌肉组织。这个问题以前已经浸泡在盐水和干燥不足或当肌肉一直在直接接触与华侨城的安装媒体在切片（ 图3B，3E）的部位肌肉组织中最常遇到的。虽然与华侨城一些接触通常是不可避免的作为安装过程的结果，应尽一切努力避免将组织学评价和OCT用于安装区域之间的接触。与此相反，用-80℃的冰箱中（ 图3C）和液氮（ 图3D）冻结的样品提供示例s表示是次优的温度冻结或速度产生冻结神器。通过将组织在-80℃冰箱中遇到的慢速冷冻过程提供冷冻神器的一个极端的例子。当使用液态氮，其主要问题是，液态氮和组织之间的接触导致气态氮气鞘，以形成在所述组织液界面，而这种气体的界面是不足够冷，以产生快速冷冻肌肉1。这可以产生显著冷冻工件试样的表面附近，但是试样的内部区域更可能闪冻在适当的速率，并且可以完全幸免冷冻工件。而在液氮肌肉组织的简单的浸泡是隔靴搔痒的异戊烷冷冻过程一样有用，它可以为大肌肉标本的情况下，冻结，其中异戊烷是不可用的（如临床尸检标本的机构是有用的出临床肌肉病理实验室）。使用这种策略的结果通常是更好地利用组织的大部分时（&gt; 2cm的几个维度）。 而避免冷冻工件提供了最佳的组​​织学结果，这种技术也得到了发展（和本文中所描述的），其基本上反转冻结的工件，以允许未正确冷冻组织的评估。组织被不正当地冻结（ 图3E）可以被融化并严格控制的条件下（ 图3F）下再冷冻，以允许水在纤维中的重新分布，形态和保存是可获得的程度可以是优秀的。根据我们的经验，这个过程会保留在组织的细胞内的形态，以显着的程度，但是它经常会产生纤维的肌内膜空间内的伪迹的分离。因为许多病理端点被发现在细胞内空间以及在在肌内膜空间，最好用特殊染色观察（或者突出纤维化或物色其他细胞类型），这些伪迹的变化是不可能真正削弱肌肉的病理评价。 <img alt="图1" fo:content-width="5in" src="/files/ftp_upload/51586/51586fig1highres.jpg" width="500" /> 图1。分流的组织为骨骼肌的结构，功能和细胞的研究。根据所希望的研究类型，收集骨骼肌组织可以以各种不同的方式获得最佳的结果进行处理。本文概述的协议描述了会审或处理骨骼肌标本进行异地的研究，以优化结果从专家的核心设施获得的方法。 <img alt="图2" fo:content-width="5in" src="/files/ftp_upload/51586/51586fig2highres.jpg" wi嗞嗞="500"/&gt; 图2。用于肌肉冷冻装置的实施例，由于在协议中所述，最佳肌肉冷冻需要在肌肉组织中的异戊烷是在其凝固温度的浸泡。获得冰冷异戊烷的一种安全和有效的方法包括使用一金属容器的被冷却的周围杜瓦含有液氮的异戊烷。因为它往往是方便的双手自由的，它可以使用一个括号，如环站（A）来保存异戊烷容器中的液氮，同时防止两种液体的混合物。或者，所述容器也可以在手动液氮（B）的保持。在任一情况下，该组织不应被放入异戊烷，直到异戊烷是明显地冻结在容器（通常为不透明的白色鹅卵石）的底部，然后应浸泡10-20秒，以允许完全冻结。 ISOPEntane在冷冻温度下产生最小的"雾"，（ 即 ，温度不够，如果相当多雾的话）。一旦组织被冻结，（直，然后进入冷却器或冷冻库），直接将其放入预先冷却的容器中，以防止意外的解冻冷冻后。 （三）适当安装冷冻肌肉应该有大部分的肌肉组织冷冻华侨城的一个碱基突出。 <img alt="图3" fo:content-width="5in" src="/files/ftp_upload/51586/51586fig3highres.jpg" width="500" /> （ 一 ）适当的异戊烷冷冻肌肉图3。冻结的有效解冻/再冻结神器，和的例子。代表性的图像，（B）肌肉中异戊烷冻结适当的，但同时在与华侨城的安装介质接触，（C）肌肉冻结在-80℃下冷冻，和（D </STRONG&gt;）的肌肉在液氮中冷冻，所有这些都显示不同程度的冻结神器。如图（B），华侨城的高液体含量产生显著冻结神器肌肉是与它接触，应采用华侨城需要安装所以只有少量，和肌肉的组织学评价应该是"站立出"，从它的表面。使用（C）-80°C冰箱或（D）液态氮（相对于在冰冷的异戊烷冻结的速度）冻结过程的缓慢会导致生产显著冻结神器。板E和F显示肌肉从即得（E）的初始次优的冷冻（由于与十月过度接触），然后（F）解冻，并使用本文描述的技术再冷冻同一标本。虽然人们可以观察到纤维之间的一些伪迹分离再冷冻样品中，改善我n中肌纤维细胞内的形态是显而易见的。 <a href="https://www.jove.com/files/ftp_upload/51586/51586fig3highres.jpg" target="_blank">请点击这里查看这个数字的放大版本。</a>

Watch this Scientific Journal Video about Tissue Triage and Freezing for Models of Skeletal Muscle Disease at JoVE.com

Tissue Triage and Freezing for Models of Skeletal Muscle Disease

The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

组织分流和冻结的骨骼肌疾病模型

Skeletal muscle is a unique tissue because of its structure and function, which requires specific protocols for tissue collection to obtain optimal ...

tissue-triage-and-freezing-for-models-of-skeletal-muscle-disease

Research

JoVE Journal

Biology

39.9K 次观看.  Medical College of Wisconsin. The analysis of skeletal muscle tissues to determine structural, functional, and biochemical properties is greatly facilitated by appropriate preparation. This protocol describes appropriate methods to prepare skeletal muscle tissue for a broad range of phenotyping studies.

视频: Tissue Triage and Freezing for Models of Skeletal Muscle Disease

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 ...

科研

生物学

Purification of Progenitors from Skeletal Muscle

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing in a "satellite cell position" between the myofiber plasma membrane and the laminin-rich basement membrane that ensheaths it, are self-renewing cells that are solely committed to the myogenic lineage1,2. We have recently described a second class of vessel associated progenitors that can generate myofibroblasts and white adipocytes, which responds to damage by efficiently entering proliferation and provides trophic support to myogenic cells during tissue regeneration3,4. One of the most trusted assays to determine the developmental and regenerative potential of a given cell population relies on their isolation and transplantation5-7. To this end we have optimized protocols for their purification by flow cytometry from enzymatically dissociated muscle, which we will outline in this article. The populations obtained using this method will contain either myogenic or fibro/adipogenic colony forming cells: no other cell types are capable of expanding in vitro or surviving in vivo delivery. However, when these populations are used immediately after the sort for molecular analysis (e.g qRT-PCR) one must keep in mind that the freshly sorted subsets may contain other contaminant cells that lack the ability of forming colonies or engrafting recipients.

Method for the enzymatic dissociation, surface labeling and purification by flow cytometry of fibro/adipogenic and myogenic progenitors from murine skeletal muscle.

纯化的骨骼肌的先觉者

Skeletal muscle contains multiple progenitor populations of distinct embryonic origins and developmental potential. Myogenic progenitors, usually residing ...

Procedures for Rat in situ Skeletal Muscle Contractile Properties

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal circulation, intact whole muscle, at body temperature. This includes the study of contractile responses like posttetanic potentiation, staircase and fatigue. Furthermore, the consequences of disease, disuse, injury, training and drug treatment can be of interest. This video demonstrates appropriate procedures to set up and use this valuable muscle preparation.
To set up this preparation, the animal must be anesthetized, and the medial gastrocnemius muscle is surgically isolated, with the origin intact. Care must be taken to maintain the blood and nerve supplies. A long section of the sciatic nerve is cleared of connective tissue, and severed proximally. All branches of the distal stump that do not innervate the medial gastrocnemius muscle are severed. The distal nerve stump is inserted into a cuff lined with stainless steel stimulating wires. The calcaneus is severed, leaving a small piece of bone still attached to the Achilles tendon. Sonometric crystals and/or electrodes for electromyography can be inserted. Immobilization by metal probes in the femur and tibia prevents movement of the muscle origin. The Achilles tendon is attached to the force transducer and the loosened skin is pulled up at the sides to form a container that is filled with warmed paraffin oil. The oil distributes heat evenly and minimizes evaporative heat loss. A heat lamp is directed on the muscle, and the muscle and rat are allowed to warm up to 37&deg;C. While it is warming, maximal voltage and optimal length can be determined. These are important initial conditions for any experiment on intact whole muscle. The experiment may include determination of standard contractile properties, like the force-frequency relationship, force-length relationship, and force-velocity relationship.
With care in surgical isolation, immobilization of the origin of the muscle and alignment of the muscle-tendon unit with the force transducer, and proper data analysis, high quality measurements can be obtained with this muscle preparation.

Procedures for Rat in situ Skeletal Muscle Contractile Properties

This video demonstrates the surgical preparation and procedures needed to study the contractile responses of the rat medial gastrocnemius muscle preparation in situ. This preparation allows measurement of skeletal muscle contractile properties under physiological conditions. The animal is anesthetized and the muscle is separated from surrounding tissue at its distal end. The Achilles tendon is attached to a force transducer, allowing measurement of the muscle&#x2019;s contractile response at 37 degrees C with an intact circulation.

对大鼠的过程<em在原位</em骨骼肌收缩性能

There are many circumstances where it is desirable to obtain the contractile response of skeletal muscle under physiological circumstances: normal ...

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 ...

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 Blood-vessel-derived Multipotent Precursors from Human Skeletal Muscle

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective isolation in culture. Recently, using fluorescence-activated cell sorting (FACS), we and other researchers prospectively identified and purified three subpopulations of multipotent precursor cells associated with the vasculature of human skeletal muscle. These three cell populations: myogenic endothelial cells (MECs), pericytes (PCs), and adventitial cells (ACs), are localized respectively to the three structural layers of blood vessels: intima, media, and adventitia. All of these human blood-vessel-derived stem cell (hBVSC) populations not only express classic MSC markers but also possess mesodermal developmental potentials similar to typical MSCs. Previously, MECs, PCs, and ACs have been isolated through distinct protocols and subsequently characterized in separate studies. The current isolation protocol, through modifications to the isolation process and adjustments in the selective cell surface markers, allows us to simultaneously purify all three hBVSC subpopulations by FACS from a single human muscle biopsy. This new method will not only streamline the isolation of multiple BVSC subpopulations but also facilitate future clinical applications of hBVSCs for distinct therapeutic purposes.

Blood vessels within human skeletal muscle harbor several multi-lineage precursor populations that are ideal for regenerative applications. This isolation method allows simultaneous purification of three multipotent precursor cell populations respectively from three structural layers of blood vessels: myogenic endothelial cells from intima, pericytes from media, and adventitial cells from adventitia.

从人骨骼肌隔离血管源性多能前体

Since the discovery of mesenchymal stem/stromal cells (MSCs), the native identity and localization&#160;of MSCs have been obscured by their retrospective ...

Isolation and Characterization of Exosomes from Skeletal Muscle Fibroblasts

Exosomes are small extracellular vesicles released by virtually all cells and secreted in all biological fluids. Many methods have been developed for the isolation of these vesicles, including ultracentrifugation, ultrafiltration, and size exclusion chromatography. However, not all are suitable for large scale exosome purification and characterization. Outlined here is a protocol for establishing cultures of primary fibroblasts isolated from adult mouse skeletal muscles, followed by purification and characterization of exosomes from the culture media of these cells. The method is based on the use of sequential centrifugation steps followed by sucrose density gradients. Purity of the exosomal preparations is then validated by western blot analyses using a battery of canonical markers (i.e., Alix, CD9, and CD81). The protocol describes how to isolate and concentrate bioactive exosomes for electron microscopy, mass spectrometry, and uptake experiments for functional studies. It can easily be scaled up or down and adapted for exosome isolation from different cell types, tissues, and biological fluids.

This protocol illustrates the 1) the isolation and culture of primary fibroblasts from the adult mouse gastrocnemius muscle as well as 2) purification and characterization of exosomes using a differential ultracentrifugation method combined with sucrose density gradients followed by western blot analyses.

骨骼肌成纤维细胞外泌体的分离和表征

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

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

We present protocols for three different methods for the homogenization of four different muscle groups of rat skeletal muscle tissue to measure and compare the levels of nitrate and nitrite. Furthermore, we compare different sample weights to investigate whether tissue sample size affects the results of homogenization.

用于硝酸盐和亚硝酸盐测量的大鼠骨骼肌匀浆的制备

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions ...

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Transient gene expression modulation in murine skeletal muscle by plasmid electroporation is a useful tool for assessing normal and pathological physiology. Overexpression or knockdown of target genes enables investigators to manipulate individual molecular events and, thus, better understand the mechanisms that impact muscle mass, muscle metabolism, and contractility. In addition, electroporation of DNA plasmids that encode fluorescent tags allows investigators to measure changes in subcellular localization of proteins in skeletal muscle in vivo. A key functional assessment of skeletal muscle includes the measurement of muscle contractility. In this protocol, we demonstrate that whole muscle contractility studies are still possible after plasmid DNA injection, electroporation, and gene expression modulation. The goal of this instructional procedure is to demonstrate the step-by-step method of DNA plasmid electroporation into mouse skeletal muscle to facilitate uptake and expression in the myofibers of the tibialis anterior and extensor digitorum longus muscles, as well as to demonstrate that skeletal muscle contractility is not compromised by injection and electroporation.

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in mice.