Name: immunolabelling myofiber degeneration in muscle biopsies
Uploaded: 2019-12-05T12:30:00
Description: Watch this Scientific Journal Video about Immunolabelling Myofiber Degeneration in Muscle Biopsies at JoVE.com

这项工作得到了法国与异体病协会的支持。作者感谢佩拉·雷耶斯-费尔南德斯博士和马修·博罗克博士认真阅读手稿。

实验是根据法国和欧洲共同体的立法（许可证号11-00010）进行的。
1. 特拉加坎特牙龈制备
<ol><li>在玻璃烧杯中，将6克的龙甘粉溶解在100 mL的去离子水中。用铝箔盖住，离开至少3小时。偶尔用金属铲子手动搅拌，因为混合物迅速变得粘稠。</li>
	<li>将酒水混合物放在60°C下过夜，放在水浴或烤箱中。小心密封烧杯，避免干燥。在等量报价之前至少搅拌一次。</li>
	<li>在4°C下储?...

<ol>
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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=Necroptosis+mediates+myofiber+death+in+dystrophin-deficient+mice.">Necroptosis mediates myofiber death in dystrophin-deficient mice.</a> Nature Communications. 9 (1), 3655 (2018).</li><li>Moens, P., Baatsen, P. H., Marechal, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+susceptibility+of+EDL+muscles+from+mdx+mice+to+damage+induced+by+contractions+with+stretch.">Increased susceptibility of EDL muscles from mdx mice to damage induced by contractions with stretch.</a> Journal of Muscle Research and Cell Motility. 14 (4), 446-451 (1993).</li><li>Tidball, J. G., Villalta, S. 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J., Bradley, D. M., Salmon, R., Clarke, A., Hartley, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Newborn+bloodspot+screening+for+Duchenne+muscular+dystrophy:+21+years+experience+in+Wales+(UK).">Newborn bloodspot screening for Duchenne muscular dystrophy: 21 years experience in Wales (UK).</a> European Journal of Human Genetics. 21 (10), 1049-1053 (2013).</li><li>Serrano, A. L., Munoz-Canoves, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+dysregulation+of+fibrosis+in+skeletal+muscle.">Regulation and dysregulation of fibrosis in skeletal muscle.</a> Experimental Cell Research. 316 (18), 3050-3058 (2010).</li><li>Rosenberg, A. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune-mediated+pathology+in+Duchenne+muscular+dystrophy.">Immune-mediated pathology in Duchenne muscular dystrophy.</a> Science Translational Medicine. 7 (299), (2015).</li><li>Kobayashi, Y. M., Rader, E. P., Crawford, R. W., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endpoint+measures+in+the+mdx+mouse+relevant+for+muscular+dystrophy+pre-clinical+studies.">Endpoint measures in the mdx mouse relevant for muscular dystrophy pre-clinical studies.</a> Neuromuscular Disorders. 22 (1), 34-42 (2012).</li><li>Saunders, N. R., Dziegielewska, K. M., Mollgard, K., Habgood, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Markers+for+blood-brain+barrier+integrity:+how+appropriate+is+Evans+blue+in+the+twenty-first+century+and+what+are+the+alternatives?+Frontiers+in+Neuroscience.">Markers for blood-brain barrier integrity: how appropriate is Evans blue in the twenty-first century and what are the alternatives? Frontiers in Neuroscience.</a> 9. 385, (2015).</li><li>Straub, V., Rafael, J. A., Chamberlain, J. S., Campbell, K. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+for+muscular+dystrophy+show+different+patterns+of+sarcolemmal+disruption.">Animal models for muscular dystrophy show different patterns of sarcolemmal disruption.</a> Journal of Cell Biology. 139 (2), 375-385 (1997).</li><li>Pastoret, C., Sebille, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-related+differences+in+regeneration+of+dystrophic+(mdx)+and+normal+muscle+in+the+mouse.">Age-related differences in regeneration of dystrophic (mdx) and normal muscle in the mouse.</a> Muscle and Nerve. 18 (10), 1147-1154 (1995).</li><li>Villalta, S. A., Nguyen, H. X., Deng, B., Gotoh, T., Tidball, J. 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肌纤维坏死是正常肌肉创伤性运动的一个常见后果。它由本地肌肉祖孕者强大的再生能力很好地补偿。然而，在一些肌肉状况，如在MD，卫星细胞的再生能力受到慢性肌瘤病和过度纤维化的影响。最近的发现表明，肌肉纤维可以死于坏死，一种受管制的坏死形式。更具体地说，抑制肾上一代可能成为DMD治疗4的新治疗策略。调查肌肉退化障碍的细胞死亡途径需要可靠的肌肉细胞死?...

骨骼肌主要由肌肉纤维（肌纤维）组成，这些纤维负责典型的自愿收缩功能。这些细胞是多核的后线结构，支持收缩过程中发生的机械应力。肌纤维膜（沙科雷马）及其细胞外基质的结构稳定性对组织平衡至关重要。卫星细胞构成成熟骨骼肌中的主要肌肉祖体群体，在健康肌肉中处于静止状态。在肌纤维死亡后，肌肉再生由卫星细胞支持，其后是一个涉及卫星细胞活化、增殖、分化和融合的肌基程序，最终形成新的多核肌纤维。
肌纤维死亡可能发生在多种肌肉条件，包括机械创伤，缺血再灌注损伤，或肌肉营养不良，它与死细胞1，2的坏死形态相关。死神死亡的特点是血浆膜的快速渗透和细胞外腔室细胞含量的释放3。它可能导致一个不受管制的过程，没有适当的细胞信号（即意外坏死），或经过编排的细胞内通路（即调节坏死）。在肌纤维中，调节的4和未调节的5个过程都可能导致坏死。阴囊炎的典型后果是释放损伤相关分子模式，激活强大的炎症反应6。在7号受伤后，在48小时和72小时观察到巨噬细胞的存在。除了在清除坏死碎片中的作用，它们在肌肉再生8，9也很重要。
肌肉萎缩症（MDs）是一个异质的病理学组，通常由沙科勒马结构的缺陷引起。杜琴肌肉萎缩症（DMD）是一种青少年X相关疾病，影响全球每3，500名男性出生10个，并且是由沙眼缺乏肌营养不良素表达引起的。DMD男孩肌肉组织的慢性退化会导致极度肌肉无力和过早死亡。坏死引起的炎症增强细胞毒性，促进肌肉纤维化和肌肉功能丧失11，12。目前针对肌肉退行性疾病根源的临床试验，如基因治疗，有望缓解肌功能障碍。因此，需要简单的技术来准确量化肌肉退化。
一些方法通常用于监测体内肌纤维损失。测量血液中肌酸激酶 （CK） 的酶活性，可以可靠地定量肌肉和心脏组织中持续的坏死。原位，血氧林和欧辛（H&E）染色是目前用于诊断评估变性再生重塑的最流行的方法。然而，死细胞H&E标签的分子基础仍不清楚。此外，表明H&E染色中肌纤维死亡的色彩修饰相对微妙，不利于可靠和可重复的定量。揭示DNA分块的方法，如端溶脱氧核糖核酸酶转移酶dUTP刻末标签（TUNEL），不完全标称坏死死亡3。它们也不太适应监测同步细胞（如肌纤维）的死亡。注射重要的染料，如埃文的蓝色染料（EBD），是评估已经失去沙科雷马完整性的肌纤维的有用替代品，但在某些实验协议中不一定方便。例如，血液样本中EBD的存在会影响CK测量的结果，这是一种色度测定。此外，细胞内对EBD的接受使得共免疫标签具有挑战性。因此，允许直接标记患有坏死的肌纤维的替代方法是值得关注的。
重要染料的作用机制依赖于坏死肌纤维中血浆膜完整性的丧失和注射染料的被动接受。同样，坏死肌纤维摄入血液蛋白，如白蛋白，其中EBD具有很强的亲和力13，14，免疫球蛋白G（IgG）和IgM15。因此，肌纤维中血液蛋白的异常存在为原位肌骨细胞分不位提供了方便的标记物。染色这些蛋白质可以是使用重要染料的替代方法。
通过使用IgG的接受作为原位肌性骨症的标记，此方案用于评估mdx营养不良症小鼠的tibialis前（TA）的肌肉退化。与替代方法技术不同，该方法具有显著的优势：1）其可重复性和执行简单性;2） 在肌肉收集之前不需要任何动物治疗，如注射循环的重要染料，以及3）作为任何常规免疫标签，它与共标签兼容。

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Circular cork disks</td>
			<td>Pyramid Innovation</td>
			<td>R30001-E</td>
			<td>Don&#39;t forget to clearly label the cork so that the the ID of the sample can be determined after freezing</td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050 sn34</td>
			<td>Muscle cryosectionning should be performed between -20 and -25&#176;C. Thickness:7-10 micrometers.</td>
		</tr>
		<tr>
			<td>Dakopen</td>
			<td>Dako</td>
			<td>S2002</td>
			<td>A hydrophobic barrier around the muscle sections. It prevents the dispertion of medium during incubation</td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>FST</td>
			<td>91117-10</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Goat anti-Mouse IgG (H+L) antibody, Alexa Fluor Plus 488</td>
			<td>ThermoFischer Scientific</td>
			<td>A-11029</td>
			<td>Dilution: 1/500</td>
		</tr>
		<tr>
			<td>Goat anti-Rabbit IgG (H+L) antibody Alexa Fluor Plus 594</td>
			<td>ThermoFischer Scientific</td>
			<td>A32740</td>
			<td>Dilution: 1/500</td>
		</tr>
		<tr>
			<td>Goat serum</td>
			<td>Jackson ImmunoResearch</td>
			<td>005-000-121</td>
			<td>At the blocking step, use 10% dilution in PBS. For antibodies incubation, use 5% dilution in PBS</td>
		</tr>
		<tr>
			<td>Isopentane</td>
			<td>Sigma Aldrich</td>
			<td>78-78-4</td>
			<td>Freezing medium. Should be cooled down in a beaker placed in liquid nitrogen.</td>
		</tr>
		<tr>
			<td>mdx mouse</td>
			<td>Jackson Laboratory</td>
			<td>C57BL/10ScSn-Dmdmdx/J</td>
			<td>Mdx mice are mutated for the dystrophin gene. From three weeks of age, muscles are characterized by chronic degeneration</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>Zeiss</td>
			<td>Imager.D1</td>
			<td>/</td>
		</tr>
		<tr>
			<td>OCT</td>
			<td>Cellpath</td>
			<td>KMA-0100-00A</td>
			<td>Embedding matrix</td>
		</tr>
		<tr>
			<td>PFA (Paraformaldehyde)</td>
			<td>ThermoFischer Scientific</td>
			<td>28908</td>
			<td>Used for fixing cryosection (2% or 4% PFA can be used)</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Eurobio</td>
			<td>CS3PBS00-01</td>
			<td>Dilution medium for immunolabeling</td>
		</tr>
		<tr>
			<td>Precision scisors</td>
			<td>FST</td>
			<td>fst 14001-12 and fst 14001-14</td>
			<td>Used for the muscle collection</td>
		</tr>
		<tr>
			<td>Rabbit antibody to mouse pan-Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L9393</td>
			<td>Dilution: 1/1000</td>
		</tr>
		<tr>
			<td>Tragacanth</td>
			<td>Sigma Aldrich</td>
			<td>G1128</td>
			<td>Aliquots to keep at +4&#176;C</td>
		</tr>
	</tbody>
</table>

immunolabelling myofiber degeneration in muscle biopsies

肌肉纤维坏死（肌骨病）在多种肌肉状况的发病机制中起着核心作用，包括肌肉萎缩症。解决肌肉营养不良发病机制原因的治疗方案有望缓解肌肉退化。因此，需要一种方法来测定和量化肌肉活检中细胞死亡的程度。观察原位肌纤维退化的常规方法要么定量不足，要么依靠注射重要染料。本文描述了免疫荧光方案，通过靶向肌纤维的免疫球蛋白G（IgG）的摄入来染色坏死肌纤维。IgG的取法基于细胞特征特征，包括1）血浆膜完整性的丧失与损伤相关的分子模式的释放，以及2）血浆蛋白的接受。在鼠阵横截面中，肌纤维、细胞外基质蛋白和小鼠IgG的共免疫标签允许对肌纤维进行清洁和直接的识别，并造成坏死的命运。这种简单的方法适用于定量分析，适用于所有物种，包括人类样品，并且不需要注射重要染料。IgG吸收对坏死肌纤维的染色也可以与其他共免疫标签搭配使用。

肌纤维被含有层宁的细胞外基质包围。红色染色划定了肌纤维的外围，并允许其识别。IgG 显示为绿色。核被DAPI染色，在显微镜下被发现为蓝色。然而，核在这里以白色显示（图1）。弱IgG免疫反应预计在细胞外腔，在炎症的情况下可以增加。肌纤维内的绿色染色反映了IgG的存在。
Mdx 小鼠 TA 的特点是在 3 ...

Watch this Scientific Journal Video about Immunolabelling Myofiber Degeneration in Muscle Biopsies at JoVE.com

Immunolabelling Myofiber Degeneration in Muscle Biopsies

这里描述的是肌肉冷冻科坏死肌纤维直接免疫标签的协议。坏死细胞可渗透到血清蛋白，包括免疫球蛋白G（IgG）。揭示肌纤维对IgG的摄取，允许识别和定量肌纤维，无论肌肉状况如何，都会发生坏死。

肌肉活检中的免疫标记肌纤维退化

The necrosis of muscle fibres (myonecrosis) plays a central role in the pathogenesis of several muscle conditions, including muscular dystrophies. ...

Myofiber 坏死是多种神经肌肉疾病的特征，在发病机制中起着核心作用。评估肌肉退化的一种具体、可靠的方法对于诊断和转化研究非常重要。这种极其适应性强的技术允许在同一部分中同时标记多个抗原。这是有可能与重要的染料。H&E 染色是一种经验方法，不能适应可靠和可重复的结合。然而，IgG吸收染色是特异性坏死肌肉细胞，适用于人类活检。这种技术可以提供洞察所有神经肌肉疾病的特点是 myonecrosis 。从感兴趣的腿上取下头发后，将四周大的雄性 mdx 动物放在上角位置，将爪子固定到软木板上。使用精密剪刀或钳子将腿部皮肤从脚到膝盖去除，以暴露头骨和头骨前部的整个长度。使用剪刀在头骨和头骨前做切口。使用钳子小心地从肌肉表面去除表皮层。在脚踝，使用钳子将头骨前肌腱与其他肌腱分离，然后轻轻地拉上肌腱，将其与头骨和周围肌肉隔离。当头骨前腹部完全分离时，将肌腱向上，使只有头骨前肌的近部部分附着在膝盖上。轻轻地切断近向肌腱尽可能接近的帕泰拉。将头骨放在用盐水轻轻打湿的纱布上，以避免干燥。将含有70至100毫升异丙烷的烧杯部分浸入液氮容器中，直到烧杯底部的异丙烷变成白色固体。仔细预先标记软木塞的另一侧，以便冷冻后能正确识别活检，并在 20 比 11 到 8 毫米圆形软木塞上添加约 0.5 毫升的曲糖胶。使用钳子将牙龈前肌嵌入牙龈肌腱侧向上。用垂直于软木表面的轴抓住肌肉。牙龈前部至少一半的肌腱侧应保持未被牙龈覆盖。然后迅速将软木塞与嵌入的肌肉浸入冷藏、未冻结的异丙烷中约两分钟，以便完全冻结。获取冷冻组织部分，将低温温度设置为零下 25 摄氏度，并使用最佳切割温度化合物将组织固定到低温热器切割块上。修剪样品，直到达到肌肉腹部。获取 7 到 10 微米部分，收集每个玻璃幻灯片至少两个节，在捕获这些部分时将幻灯片置于室温下。在通风环境中，让组织在室温下干燥至少 20 分钟。然后将部分储存在零下 80 摄氏度，直到它们染色。免疫标签组织。首先，在室温下在通风环境中解冻幻灯片至少15分钟。接下来，使用疏水笔描绘各部分。在潮湿的腔室中用2%的甲醛固定组织10分钟。在固定结束时，每次洗涤用两次 5 分钟洗涤，冲洗部分。用在PBS中稀释的10%山羊血清阻止任何非特异性结合。室温1小时后，用感兴趣的原抗体标记各部分，在室温下在5%山羊血清中稀释2小时。在孵育结束时，在PBS中用3个5分钟的洗涤液冲洗幻灯片，然后用适当的荧光结合二次抗体标记，在室温下保持45分钟，防止光线。在孵育结束时，用三次10分钟的PBS洗涤滑梯。使用荧光安装介质安装该部分，该介质包含每毫升 DAPI 100 毫微克和盖滑。然后在成像前将幻灯片在摄氏四度下干燥。来自四周大 mdx 小鼠的 Tibialis 前肌肉通常显示异质轮廓，包括在同一横截面中退化和再生区域影响不佳的区域。不受轻度影响肌肉区域的影响，含有大而均匀的肌黄体，低密度的核主要位于边缘或肌骨之间。IgG阳性 myofibers 在此类健康或轻度受影响的区域通常不存在，而 Iofibers 内的 IgG 免疫反应性表示 myofibers。新形成的肌纤维在再生的早期阶段以小尺寸存在，随着肌肉祖细胞的融合，逐渐扩大，从而有助于同步细胞。在这个过程中，肌黄体留在再生组织的中心，有一簇小的，集中核的肌黄玉，指示最近再生的肌黄体，为了尽量减少神器的风险，重要的是保持肌肉直，当冻结组织和在正确的温度。异丙烷和PFA应始终小心处理，并在烟罩下处理。

Research

JoVE Journal

Biology

Preparation and Using Phantom Lesions to Practice Fine Needle Aspiration Biopsies

制备及使用幻影病变实践细针穿刺活检

Currently, health workers including residents and fellows do not have a suitable phantom model to practice the fine- needle aspiration biopsy (FNAB) ...

科研

生物学

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

体外评估对离体骨骼肌的收缩，疲劳性和交替“

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

Evaluation of Muscle Function of the Extensor Digitorum Longus Muscle Ex vivo and Tibialis Anterior Muscle In situ in Mice

伸趾长肌的肌肉功能评价<em&gt;体外</em和胫骨前肌<em在原位</em在小鼠

Body  movements are mainly provided by mechanical function of skeletal muscle. Skeletal  muscle is composed of numerous bundles of myofibers that are ...

Generation of Myospheres From hESCs by Epigenetic Reprogramming

Myospheres的产生从人类胚胎干细胞通过表观遗传修饰

Generation of a homogeneous and abundant population of skeletal muscle cells from human embryonic stem cells (hESCs) is a requirement for cell-based ...

Methylnitrosourea (MNU)-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Methylnitrosourea MNU-induced Retinal Degeneration and Regeneration in the Zebrafish: Histological and Functional Characteristics

Methylnitrosourea（MNU）诱导的视网膜变性和再生的斑马鱼：组织学和功能特征

Retinal degenerative diseases, e.g. retinitis pigmentosa, with resulting photoreceptor damage account for the majority of vision loss in the industrial ...

Detection of Tissue-resident Bacteria in Bladder Biopsies by 16S rRNA Fluorescence In Situ Hybridization

16S rRNA 荧光原位杂交检测膀胱活检中的组织驻留细菌

Visualization of the interaction of bacteria with host mucosal surfaces and tissues can provide valuable insight into mechanisms of pathogenesis. While ...

Isolating Myofibrils from Skeletal Muscle Biopsies and Determining Contractile Function with a Nano-Newton Resolution Force Transducer

使用纳米-牛顿分辨率力转换器从骨骼肌肉活检中隔离肌异物并确定收缩函数

Striated muscle cells are indispensable for the activity of humans and animals. Single muscle fibers are comprised of myofibrils, which consist of ...

Isolation of Human Ventricular Cardiomyocytes from Vibratome-Cut Myocardial Slices

人体心室心肌细胞与颤音-切切心肌切片分离

The isolation of ventricular cardiac myocytes from animal and human hearts is a fundamental method in cardiac research. Animal cardiomyocytes are commonly ...

Collection of Skeletal Muscle Biopsies from the Superior Compartment of Human Musculus Tibialis Anterior for Mechanical Evaluation

从人类肌肉提比利斯前部高级隔间收集骨骼肌肉活检，用于机械评估

The mechanical properties of contracting skeletal fibers are crucial indicators of overall muscle health, function, and performance. Human skeletal muscle ...

Single Myofiber Culture Assay for the Assessment of Adult Muscle Stem Cell Functionality Ex Vivo

单一肌纤维培养测定法用于评估体外成人肌肉干细胞功能

Adult skeletal muscle tissue harbors a stem cell population that is indispensable for its ability to regenerate. Upon muscle damage, muscle stem cells ...