We thank the Animal House and the Integrated Microscopy Facilities of IGB-CNR. This work has benefited from research funding from the European Community's Seventh Framework Programme in the project ENDOSTEM (Activation of vasculature associated stem cells and muscle stem cells for the repair and maintenance of muscle tissue, grant agreement number 241440), the Italian Ministry of Education-University-Research (MIUR-PRIN2 010-2011) to G.M. and S.B. and PON Cluster IRMI to G.M., and the CARIPLO foundation to G.M. and S.B.

所有实验均严格按照动物研究机构的指导方针进行，并按照有关动物实验的法律公共卫生，动物健康，营养和健康的意大利卫生部食品安全主管部门批准。颈椎脱位，程序可以根据IACUC或等值要求各不相同，从机构的机构。 1.心脏毒素注射在胫骨前肌<ol><li>通过在启动过程之前，稀释在无菌磷酸盐缓冲盐水（PBS）中或在水中的心脏毒素原液制备10μM的工作心脏毒素（CTX）的溶液中。 注意:CTX溶于水以1毫克/毫升。制备70微米原液。分装和储存在-20°C。用于该协议CTX是具有大约7 000道尔顿的分子量cardiotoxins的混合物。 注意:建议穿双层手套，口罩的卫生和安全眼镜（或者安全眼镜，建议罩下工作），并要非常小心，以避免与解决任何联系，即使稀释。 </li><li>麻醉氯胺酮和赛拉嗪（80毫克/公斤和10毫克/体重kg）或其它经批准的可用麻醉剂的腹膜内注射小鼠。 </li><li>用鼠标面朝上，喷后肢用70％的乙醇。可视化的胫骨前（TA）肌肉的准确位置，用手术刀的帮助下，切割毛发走在小腿的前部，膝盖下。 注意:TA肌肉源于胫骨的近侧主体，下降横向于骨，并在脚踝结束。其长肌腱伸入穿过脚踝脚（ 见图1A）。 </li><li>通过拉动柱塞背面慢慢绘制〜100μL的该溶液放入注射器。 </li><li>去除气泡，直立持注射器（与针向上）。轻拉活塞，然后敲击注射器一边帮附着在管的内部的气泡来向上。慢慢地按下柱塞释放出大量气泡。液体滴几滴将走出针。在这一点上，请务必重新挑液体成管，而不是分散的，给予心脏毒素的毒性。 </li><li>撤回溶液进入注射器，直至达到所需的剂量。注射的每个TA肌肉CTX溶液20微升。 </li><li>由以下的胫骨为指导的位置，从朝向膝盖腱插入在TA的中心针。由于TA是一个薄组织，进行注射足不出户过深（插入针2/3毫米深，在大约10°到20°角），避免超越肌肉本身（ 图1A）。 </li><li>不要拔出针头在注射后立即，但等待2 - 3秒，以防止泄漏CTX的年龄从肌肉下降。 </li><li>在手术后，放置在加热板（在37℃）的笼子，直到小鼠是清醒的，因为麻醉剂和所用的乙醇降低体温。 </li></ol> 2.胫前隔离注意:肌肉可以在不同的时间点心脏毒素注射后根据实验要求中分离出来。 <ol><li>颈椎脱位牺牲鼠标。 </li><li>喷用70％乙醇的后肢。在实验过程中，动物可固定到支撑保持下来。 </li><li>切在近端肌腱（脚以上）的电平的皮肤，插入皮肤下剪刀的刀片之一，并切开皮肤向上，在膝盖的方向。随着产钳的帮助下，拉起皮肤完全暴露的肌肉。小心去除所有的皮肤和从感兴趣的区域中的头发。 </li><li>消除慢慢捏或切割筋膜 （与日在尖镊子或细微的胫骨（ 图1B）的相对侧解剖剪刀）肌肉的极端周边。一旦打破，温和地去除薄尖镊子的帮助下筋膜 ;避免损坏下面的肌肉。 注意:与其他肌肉和器官中，TA由筋膜 （腿的深筋膜），结缔组织的薄片是位于深皮肤下，并需要被除去以容易分离下面的肌肉覆盖。 </li><li>可视化远端TA肌腱和伸指肌腱的脚上面，这是并列的。趾长伸肌（EDL）肌的腱位于略低于TA肌腱。成年小鼠的筋是肉眼可见的。 </li><li>除去以下以下两种技术中的一种对TA肌肉: <ol><li>切细微解剖剪刀既筋，和他们拉向近侧，牵着摹用钳子筋。分开的两个筋，在相反的方向轻轻拉动它们的肌肉分开。 </li><li>薄尖镊子的帮助下，通过将腱本身（ 图1C，上图）所示的尖端分离从伸指肌腱远端TA肌腱。轻轻滑动的TA低于镊子将其从下面的肌肉（ 图1C，底部图片）分开。 </li><li>保持肌肉下用镊子把它微微抬起，切TA肌腱，轻轻地向上拉，直到只有膝盖以下附后（ 图1D，左图）的区域。切TA膝盖以下，之后用剪刀（ 图1D，右图）肌肉的边缘。 注意:如果肌肉保持小幅连接到两侧，用细微解剖剪刀，帮助卸下。 注意:如果镊子不要轻易适合TA下方，它是可能的筋膜尚未完全除去，它仍然是对肌肉的顶部可见。在这种情况下，继续彻底删除前筋膜 。 </li></ol></li></ol> 3.新鲜冰冻肌肉技术<ol><li>放置黄蓍胶或软木的切片的类似粘合剂冷冻化合物（ 例如，最佳切削温度（OCT）化合物）的量小。冷冻前标签在反面的软木塞的切片，如果需要的话（ 图2A）。 </li><li>为了获得肌肉的横截面，水槽的TA进入黄蓍胶通过插入远端肌腱和外而使肌肉的约3/4，并确保有一个垂直位置的肌肉相对于所述软木塞（ 图2A）。 </li><li>填充的铝罐（可选地，一个金属杯或玻璃烧杯），用异戊烷。暂停异戊烷的可以含有杜瓦LIQUIð氮，不允许液氮进入罐。 注意:异戊烷需要达到适当的温度（-150〜-160℃）的组织的最佳的冻结。当一些白色固体颗粒形成在罐的底部和异戊烷变得略微粘稠达到正确的温度。 注:固体颗粒形成之前不要冷冻样品，因为它可以导致冻结文物。另一方面，如果异戊烷变得完全固体，把它在室温下，直到它被解冻，确保一些白色颗粒仍然存在。 </li><li>使用Mixter镊子，迅速浸软木与肌肉入异戊烷，保持肌肉定位向下，并确保释放软木塞当它完全浸入在液体中。以这种方式，而检体浮起，只有软木的反面应在液体中可见。 </li><li>离开SAmple在异戊烷1分钟。 </li><li>使用Mixter镊子（或类似镊子），取肌肉和浸入其在液氮中至少2分钟。 </li><li>单独包装的标本在标有铝箔，并立即将它们放置在干冰（-70℃），或直接将其存储在-80℃冰箱中。 注意:在整个过程中，从步骤3.4至3.7，这是非常重要的是要保持正确的温度，并防止解冻样本（ 如 ，转移到-80℃的冰箱中）。在不受控制的解冻和水微晶是在组织内本，并最终导致工件的形成或肌纤维的击穿的再冻结的温度升高的结果（可视化的组织切片中的纤维的中心大白色空穴）。 </li></ol> 4.冷冻肌肉的低温恒温器切片<ol><li>设置低温恒温室温度tØ-20〜-22℃。 </li><li>把试样存根，刀片，并在低温恒温室中的样品，使它们平衡至少30分钟。 </li><li>放置在试样存根冷冻化合物（ 例如，OCT化合物中）少量的，并与该冷冻化合物固化前的试样浸入软木。在此期间，将在刀片保持器的刀片。 </li><li>使用所提供的螺钉来定向与该叶片夹持器（ 图2B）的试样保持器对准试样和刀片。 </li><li>切片厚度设为10微米。进行到切割组织。为驱动轮的每一匝，试样保持器前进朝向叶片的受控距离。 </li><li>放置在偏振幻灯片（每张幻灯片8至10节）（ 图2C）的部分。 </li><li>切片后，存放在幻灯片-80℃。 注:样品可以储存在-80℃冰箱和用于furthe重用- [R切片，如果妥善处理和储存。 </li></ol> 5，常规病理染色（苏木曙红渍） 注意:几个组织学染色可以在肌肉部分根据分析来进行。对于形态和形态分析的常规病理染色是苏木曙红（H＆E）染色重铬酸。苏木染色核深紫色。核染色复染与曙红（粉红/红色），其染色嗜酸性结构，例如在细胞质中的肌纤维。 <ol><li>完全风干冷冻切片为10个部分 - 在室温下15分钟，误入染色托盘中的幻灯片，并把托盘染色低谷。 </li><li>染色1分钟用苏木。 </li><li>打下相当薄弱自来水喷下的染色槽10分钟洗出苏木。 </li><li>浸入载玻片5分钟，在95％的乙醇中，如果曙红的醇溶液将使用（跳到吨他在水性曙红溶液的情况下，工序）。 </li><li>染液1分钟用曙红溶液。 </li><li>用清水冲洗，以消除过度伊红的解决方案。 注意:与任一苏木或曙红染色的最佳时机应为每个新的批次来限定。 </li><li>脱水的样品在梯度乙醇系列:50％和70％（每几秒钟）。迅速浸入在95％乙醇，随后2的变化在100％乙醇，每次5分钟。 </li><li>明确在2变化二甲苯中，每5分钟或更长的时间。这个步骤必须在化学通风橱中进行。 </li><li>装入滑动与基于二甲苯的安装平台，施加在滑动安装介质，并用盖玻片覆盖几滴。安装介质也可以应用到盖玻片和盖玻片放在滑动。 <ol><li>避免形成该载玻片和盖玻片之间的气泡。为了消除气泡，保持滑板垂直（安装后），挤出多余的MO通过在钝钳（或类似的钝工具）盖玻片轻轻按压unting介质和气泡。 </li></ol></li><li>保持滑动罩下几个小时或过夜以让二甲苯在进行分析之前完全蒸发。 </li><li>通过使用每鼠的至少一个全肌肉部的非重叠序列图像执行的H＆E染色的再生肌切片的形态和形态测定分析。捕捉图像与明场显微镜，用放大20倍。 注:建议5只小鼠的最小数量，以获得测量值的统计学意义。 注:图像分析软件可用于这一目的，比如免费下载软件ImageJ的（http://imagej.nih.gov/ij/）。 </li></ol>

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The authors have nothing to disclose.

这里，我们描述了协议来诱导骨骼肌（ 即，CTX的肌内注射）急性损伤。它被广泛地用作一种强大的工具来研究骨骼肌再生的动态体内 。 CTX注射诱导的肌纤维，它是由肌膜的去极化和纤维12的收缩而引起的变性，并触发，导致肌肉再生事件的级联。骨骼肌是在注射和损伤后所要求的时点解剖，根据实验的要求，并用于后续的组织学分析。解剖的肌肉可以被冻结，夹杂在冷冻保护媒体之后，或包括在石蜡中。然而，这些程序需要预先固定用多聚甲醛，它可以产生伪像，并且可以最终影响到随后的分析的步骤。直接冻结组织可避免此问题。值得注意的是，许多主要抗体完全或更有效地工作的新鲜冰冻切片肌肉13，这使得这个过程更可靠。的形态测定分析，通常是在时间过程实验，其允许具有相同年龄的小鼠组之间在再生过程中的差异的评价，并与特定的遗传突变和/或药物治疗进行。虽然CTX诱导损伤的协议是高度重现本身，可能的限制是由于CTX注射的操作者相关的可变性。为了克服这一限制，但优选的是，整个时程实验由相同的操作者进行。 肌肉再生的程度可以量化为健康纤维，坏死区和炎症，再生和再生区过总面积的百分比。然而，研究慢性损伤的事件，如在营养不良的mdx英里时这种方法主要是用来策，而不是在急性损伤模型。实际上，虽然营养不良的肌肉的特征在于变性和再生的异步事件，急性损伤后跟明确定义和相应的事件。这些分析，这需要的上述形态特征的经验识别的一个限制是，它们不是完全无偏的。支持实验的结论中，语形学分析应始终进一步分析补充，如特定分子标记免疫荧光分析，以支持实验的结论。因为这个原因，优选的是使用平行分析明确地解释结果。例如，再生的肌纤维被染色阳性对于在新形成的肌纤维中特异性表达的胚胎球蛋白重链（eMyHC）。因此，eMyHC染的肌纤维的定量可以用形态学分析来进行侧由端。 该CSA的分析是比较可靠的量化，可以在H＆E染色切片或与层粘连蛋白，这标志着肌纤维的染色边缘部分的肌肉或者进行;定量是使用ImageJ的相应的宏如上所述执行。在这两种情况下，它是总是需要先评估部分的质量，并排除出现变形或卷曲的组织的区域。 旁边的组织的形态学分析，H＆E染色的切片允许的其他具体特征，如纤维化和/或脂肪组织的存在的鉴定。事实上，从纤维化细胞外基质的过度积聚发生或者如果再生受损或慢性疾病14派生的。事实上，如存放在H＆E组织学染色再生肌纤维之间苍白材料外基质积聚可见。 CTX注射的多轮可用于吨Ø模仿的慢性疾病，其中肌纤维进行变性和再生和疤痕组织的积累波异常。然而，更可靠的协议可以诱导肌肉纤维化15，并且可以执行特定组织染色，以确定和量化这些结构，如马松三色或天狼星红染色。脂肪组织的形成是在H＆E染色的切片的肌纤维之间圆形白色结构，赋予脂肪组织，其通过油红染色定量的存在的重要标志清晰可见。最后，下面描述的可用于执行使用特异性抗体和协议免疫荧光分析的协议获得骨骼肌节。

哺乳动物成人骨骼肌是由多核肌细胞（肌纤维）是专门为收缩fascicules群体形成。每个肌纤维是细长的合胞体，由肌膜（质膜）和含有肌原纤维，这是由定期多次组织收缩蛋白（肌动蛋白和肌球蛋白丝）所包围。在成年生活中，在休息的条件下，骨骼肌有其肌细胞核1非常低的营业额;的确，肌细胞核，这是位于肌纤维的周围，肌膜下，在细胞周期的G0期被捕并且无法增殖1,2。 骨骼肌必须重新生成以下的损伤，组织重塑的几个事件紧密相关的互相后达到稳态的特有能力。急性损伤或外伤之后，变性诱导，随后通过再生过程涉及不同的细胞群，包括肌肉细胞的常住人口中，卫星细胞（SCS）。事实上，在没有任何环境刺激的，卫星细胞是处于静止状态，位于肌膜和基底层3,4之间的专门利基。以下内容的损伤或疾病，旺成为激活，增殖，迁移到受损区域，并最终分化，从而引发新形成肌纤维5。活化的SC建立与不同细胞群，主要炎症细胞，这是在创伤6-8的部位招募串扰。这种串扰允许细胞遵循稳压范例通过该分子信号驱动结构上的修改，最终导致动态平衡9。此外旺，炎症和间质细胞，血管生成过程，并重新支配的事件也都参与其中，以协调的方式采取行动，以修复此高度组织化和Specialized结构。 有在研究骨骼肌再生的各个方面，不仅要了解肌肉的生理机能，同时也提高需要全过程的深入了解治疗策略的极大兴趣。几个实验的方法是目前可用来研究不同细胞群体，信号通路，以及所涉及的分子机制的标识和功能。急性损伤的小鼠模型代表一个强有力的工具来研究这个过程的许多方面。不同常用的技术来诱导急性肌肉损伤允许研究遵循再生过程在体内 ，从非常早期阶段的过程中结束。这个协议描述从肌肉注射蛇毒来源的心脏毒素的（CTX），其诱导myolysis并触发再生过程中，最多的组织样本的分析的步骤。继CTX注射，MICE可以在不同的时间点，这取决于实验要求被牺牲，并且骨骼肌可以解剖并进行进一步的分析处理。最后，我们描述了组织切片的染色方案来执行形态学观察和基本定量分析。该协议允许对急性骨骼肌再生的体内以高度重现的方式10的研究。

<table border="0" cellpadding="0" cellspacing="0" width="957">
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		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Cardiotoxin from&#160;Naja mossambica mossambica</td>
			<td style="width:199px;">SIGMA ALDRICH</td>
			<td style="width:119px;">C9759</td>
			<td style="width:191px;"></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Syringe For Insulin BD Micro-Fine+ Needle 30 G X 8 mm - Da 0,3 ml</td>
			<td style="width:199px;">BD</td>
			<td style="width:119px;">324826</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Tragacanth Gum</td>
			<td style="width:199px;">MP BIOMEDICALS,LLC</td>
			<td style="width:119px;">104792</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2-Methylbutane (Isopentane)</td>
			<td>SIGMA ALDRICH</td>
			<td style="width:119px;">78-78-4.</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">OCT Killik Solution For Inclusion Cryostat</td>
			<td>Bio-optica</td>
			<td>&#160;05-9801</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Feather Microtome Blade S35</td>
			<td style="width:199px;">Bio-optica</td>
			<td style="width:119px;">&#160;01-S35</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Glass Slide Superfrost Plus</td>
			<td style="width:199px;">Menzel-Gl&#228;ser</td>
			<td style="width:119px;">09-OPLUS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Dumon #5 Mirror Finish Forceps&#160;</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">11251-23</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Scissors Straight Sharp/Sharp</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">15024-10</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Scissors Noyes Straight</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">15012-12</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Fine Iris Scissors Straight Sharp/Sharp 10,5 Cm</td>
			<td style="width:199px;">2BIOLOGICAL INSTRUMENTS</td>
			<td style="width:119px;">14094-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Eukitt</td>
			<td style="width:199px;">Bio-optica</td>
			<td style="width:119px;">09-00100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Slide Coverslip</td>
			<td style="width:199px;">BIOSIGMA</td>
			<td style="width:119px;">VBS651</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Xylene</td>
			<td style="width:199px;">SIGMA ALDRICH</td>
			<td style="width:119px;">214736</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethanol 100%</td>
			<td>sigma-Aldrich</td>
			<td>02860-2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Hematoxyline</td>
			<td style="width:199px;">J.T. BAKER</td>
			<td style="width:119px;">3873</td>
			<td></td>
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			<td height="21" style="height:21px;width:449px;">Eosin</td>
			<td style="width:199px;">SIGMA ALDRICH</td>
			<td style="width:119px;">HT110116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:449px;">Cryostat</td>
			<td style="width:199px;">LEICA</td>
			<td style="width:119px;">CM3050 S</td>
			<td></td>
		</tr>
	</tbody>
</table>

induction of acute skeletal muscle regeneration by cardiotoxin injection

骨骼肌再生是发生在成人骨骼肌中响应损伤或疾病的生理过程。急性损伤诱导的骨骼肌再生是研究参与肌肉再生以及机制和不同玩家的事件一种广泛使用的，强大的模型系统。实际上，这个过程的详细知识是为了更好地理解的病理状况导致骨骼肌变性必需的，而且它在确定新的靶向治疗策略有助于。目前的工作描述了一个详细的和可重复的协议，并通过单次肌注的心脏毒素（CTX）诱导小鼠急性骨骼肌再生。 CTX属于家庭的蛇毒毒素和导致肌纤维的myolysis，最终触发再生事件。骨骼肌再生的动力学是由肌肉切片的组织学分析评价。该协议还示出了用于解剖，冷冻，并切割该胫骨前肌，以及，被广泛用于后续形态学和形态分析例程苏木曙红染色的实验程序。

H＆E染色允许在特定时间点的再生处理的形态的骨骼肌再生过程中进行评价。 图3显示了对野生型小鼠的受伤的TA肌肉进行时间过程分析。肌肉已经分离在CTX注射后3,7，15，和30天，如在图3A中图式。 H＆E染色横截面有代表性的图片显示肌肉修复的动力随着时间的推移（ 图3B-E）。在伤后第3天，肌肉的结构体系结构被完全破坏，并且两个变性肌纤维（坏死面积）和单核细胞是清晰可见的（ 图3B）。这种异构细胞群主要由短暂扩充卫星细胞（成肌细胞），炎性细胞从血液中招募11，和间质细胞和内皮细胞的党CIPATE在再生过程，这是在损伤部位触发。在这种情况下，通过融合成肌细胞的分化生成新的再生肌纤维。再生区的主要特征是具有位于中央的细胞核和暗染色细胞质（ 图3C）小嗜碱性肌纤维的存在。在此阶段，炎症仍是可见的，尽管它逐渐减小。再生肌纤维的大小随时间增加，引起其特征在于位于中央的细胞核（ 图3D，E）的嗜酸性粒细胞再生的肌纤维。再生肌纤维是可见在再生的后期阶段，直到该过程结束。健康肌纤维出现高度有组织的并彼此接近，具有粉红色细胞质和放置在该纤维的外周，肌膜下核。这些肌纤维通常与CTX注射液和损伤的部位现在为止，他们更难得LY代表地区再生是完全明确的地方。实际上，再生纤维是由位于中央的细胞核则移动到周边损伤仅几个月后的存在表征。 染色肌肉切片的数字图像通过添加比例尺进行空间校正，这是必要通过概述的感兴趣区域的轮廓，以精确地测量的感兴趣的区域捕获。的肌纤维横截面积（CSA）的测量是可靠的形态测定分析，它被广泛用于量化小鼠不同组之间的再生趋势的差异。这种分析需要的中央核的肌纤维的区域的测量，包括至少500和至多每部1000的纤维。同时再生的平均和再生肌纤维区域和这些区域的高斯分布是再生PROC指标ESS。增加的肌纤维CSA通常具有改进的和/或加速再生反应相关联。相反，适当的再生的故障与CSA的降低相关联。这里，我们报道了肌纤维CSA分析，指示为两者的平均值（ 图3F）和肌纤维的CSA的分布（ 图3G）在不同时间点。分析亮点肌纤维区域的分布如何随时间变化，以及如何实现更大尺寸的纤维作为再生前进的过程中的CSA移位。 <img alt="图1" src="/files/ftp_upload/54515/54515fig1.jpg" /> 图1.肌注胫肌肉的分离。 A.肌内注射在胫骨前肌的心脏毒素。 B点 。去除肌肉筋膜 。白色箭头指示薄尖镊子该捏肌肉以除去第Ë 筋膜 。 ℃。顶部图为远端TA肌腱的解剖位置。底部图为如何解除TA并将其取出，避免肌肉损伤时。 ð。左图:在TA是向上拉动远端肌腱解除。白色箭头指示远端EDL肌腱。右图:通过举办用钳子肌腱，电讯管理局局长的上边缘膝盖以下切断。白色箭头表示切割部位。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54515/54515fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54515/54515fig2.jpg" /> 图2.新鲜冰冻肌肉和肌肉冷冻冷冻切片机的切片。 A.新鲜肌肉列入黄蓍胶技术。所述的TA肌肉浸渍在从肌腱的胶，与约3/4以外和保持在相对于所述软木塞一个垂直位置。为低温恒温器切片的程序B.代表性图像。软木塞置于对试样存根冷冻化合物的量小。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54515/54515fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54515/54515fig3.jpg" /> 骨骼肌再生图3.时间进程分析。急性骨骼肌损伤的2.1实验方案。 BE。心脏毒素的苏木伊红染色切片（CTX）的代表性图片损伤后 - 处理在指定天肌肉。 B点 。黑色连续线（在图像的左侧），包围一对坏死纤维，可能是由炎性细胞侵入。虚线（右侧图片的）标记浸润单核细胞的区域。与位于市中心核C.单不成熟肌纤维是由黑色圆圈表示。 DE。大嗜酸性粒细胞再生肌纤维，与位于市中心核通过黑色圆圈标记。刻度条表示100毫米。在TA肌肉部分中央核肌纤维大小值的平均楼 。值是平均值±SEM，5只小鼠/组。 G.肌纤维横截面积（CSA）分布在6,15，和CTX注射后30天。值是平均值±SEM，5只小鼠/组。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54515/54515fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection at JoVE.com

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

该原稿描述了详细的协议来诱导成年小鼠和肌肉的随后的操作，如解剖，冷冻，切割，常规染色，和肌纤维横截面积分析急性骨骼肌再生。

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

induction-acute-skeletal-muscle-regeneration-cardiotoxin

Nanyang Technological University

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Developmental Biology

21.8K 次观看.  Institute of Genetics and Biophysics "A. Buzzati Traverso", CNR. 该原稿描述了详细的协议来诱导成年小鼠和肌肉的随后的操作，如解剖，冷冻，切割，常规染色，和肌纤维横截面积分析急性骨骼肌再生。 

视频: Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

肾再生的成年斑马鱼庆大霉素性损伤

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

科研

发育生物学

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

从人类骨骼肌成肌原细胞和成纤维细胞的分离和定量免疫细胞化学表征

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

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 ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

通过子代检测改变斑马鱼致死性骨骼突变显

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and pathological processes, such as cancer and ageing. Recently, senescence has also been implicated in tissue repair and regeneration. Therefore, it has become increasingly critical to identify senescent cells in vivo. Senescence-associated &#946;-galactosidase (SA-&#946;-Gal) assay is the most widely used assay to detect senescent cells both in culture and in vivo. This assay is based on the increased lysosomal contents in the senescent cells, which allows the histochemical detection of lysosomal &#946;-galactosidase activity at suboptimum pH (6 or 5.5). In comparison with other assays, such as flow cytometry, this allows the identification of senescent cells in their resident environment, which offers valuable information such as the location relating to the tissue architecture, the morphology, and the possibility of coupling with other markers via immunohistochemistry&#160;(IHC). The major limitation of the SA-&#946;-Gal assay is the requirement of fresh or frozen samples.
Here, we present a detailed protocol to understand how cellular senescence promotes cellular plasticity and tissue regeneration in vivo. We use SA-&#946;-Gal to detect senescent cells in the skeletal muscle upon injury, which is a well-established system to study tissue regeneration. Moreover, we use&#160;IHC to detect Nanog, a marker of pluripotent stem cells, in a transgenic mouse model. This protocol enables us to examine and quantify cellular senescence in the context of induced cellular plasticity and in vivo reprogramming.

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Here we present a detailed protocol to detect both senescent and pluripotent stem cells in the skeletal muscle upon injury while inducing in vivo reprogramming. This method is suitable for evaluating the role of cellular senescence during tissue regeneration and reprogramming in vivo.

骨骼肌损伤诱导衰老和体内重编程的评价

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

X-ray Diffraction of Intact Murine Skeletal Muscle as a Tool for Studying the Structural Basis of Muscle Disease

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal muscle. Mouse skeletal muscle has been shown to produce high quality X-ray diffraction patterns on third generation synchrotron beamlines providing an opportunity to link changes at the level of the genotype to functional phenotypes in health and disease by determining the structural consequences of genetic changes. We present detailed protocols for preparation of specimens, collecting the X-ray patterns and extracting relevant structural parameters from the X-ray patterns that may help guide experimenters wishing to perform such experiments for themselves.

We present detailed protocols for performing small-angle X-ray diffraction experiments using intact mouse skeletal muscles. With the wide availability of transgenic mouse models for human diseases, this experimental platform can form a useful test bed for elucidating the structural basis of genetic muscle diseases

完整毛里丝骨骼肌的X射线衍射作为研究肌肉疾病结构基础的工具

Transgenic mouse models have been important tools for studying the relationship of genotype to phenotype for human diseases including those of skeletal ...

Isolation, Culture, Characterization, and Differentiation of Human Muscle Progenitor Cells from the Skeletal Muscle Biopsy Procedure

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle regenerative process. There are recognized challenges to working with human primary adult stem cells, particularly human muscle progenitor cells (hMPCs) derived from skeletal muscle biopsies, including low cell yield from collected tissue and a large degree of donor heterogeneity of growth and death parameters among cultures. While incorporating heterogeneity into experimental design requires a larger sample size to detect significant effects, it also allows us to identify mechanisms that underlie variability in hMPC&#160;expansion capacity, and thus allows us to better understand heterogeneity in skeletal muscle regeneration. Novel mechanisms that distinguish the expansion capacity of cultures have the potential to lead to the development of therapies to improve skeletal muscle regeneration.

We present techniques for isolating, culturing, characterizing, and differentiating human primary muscle progenitor cells (hMPCs) obtained from skeletal muscle biopsy tissue. hMPCs obtained and characterized through these methods can be used to subsequently address research questions related to human myogenesis and skeletal muscle regeneration.

从骨骼肌活检程序分离、培养、表征和分化人类肌肉祖细胞

The use of primary human tissue and cells is ideal for the investigation of biological and physiological processes such as the skeletal muscle ...

Performing Human Skeletal Muscle Xenografts in Immunodeficient Mice

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this failure is the use of inadequate laboratory models. It is challenging to model complex human diseases in traditional laboratory organisms, but this issue can be circumvented through the study of human xenografts. The surgical method we describe here allows for the creation of human skeletal muscle xenografts, which can be used to model muscle disease and to carry out preclinical therapeutic testing. Under an Institutional Review Board (IRB)-approved protocol, skeletal muscle specimens are acquired from patients and then transplanted into NOD-Rag1null IL2r&gamma;null (NRG) host mice. These mice are ideal hosts for transplantation studies due to their inability to make mature lymphocytes and are thus unable to develop cell-mediated and humoral adaptive immune responses. Host mice are anaesthetized with isoflurane, and the mouse tibialis anterior and extensor digitorum longus muscles are removed. A piece of human muscle is then placed in the empty tibial compartment and sutured to the proximal and distal tendons of the peroneus longus muscle. The xenografted muscle is spontaneously vascularized and innervated by the mouse host, resulting in robustly regenerated human muscle that can serve as a model for preclinical studies.

Complex human diseases can be challenging to model in traditional laboratory model systems. Here, we describe a surgical approach to model human muscle disease through the transplantation of human skeletal muscle biopsies into immunodeficient mice.

在免疫缺陷小鼠中表演人类骨骼肌异种移植物

Treatment effects observed in animal studies often fail to be recapitulated in clinical trials. While this problem is multifaceted, one reason for this ...