这项研究是由基督教多普勒研究基金会的支持。我们愿在奥地利维也纳医科大学从核心设施成像感谢萨宾·劳施在整个项目的支持。初级抗体通过菲诺，S.发展，从发展研究杂交瘤细胞银行获得，由美国国立卫生研究院的NICHD创建和维护在爱荷华，生物系，爱荷华市，IA大学。

包括动物个体所有程序均符合实验动物管理的原则进行所建议的FELASA 26。由维也纳医科大学与奥地利财政部研究和科学机构审查委员会之前的研究获得批准（BMWF:德国联邦附耳Wissenschaft与研究部，参考号码:BMWF-66.009 / 0222-WF / II / 3B / 2014）。 1.肌肉收获注:在之前发表的孟等人。 27，请中详细介绍肌肉标本的正确冻结。 <ol><li> 获得整个肌肉或足够的组织动物安乐死后，立即获取整个横截面。 <ol><li>从麻醉或安乐死的大鼠删除整个肌肉。删除所有的结缔组织，并与周围的镊子和剪刀的肌腱。为一个nesthesia或安乐死，遵循FELASA 28的国际准则。 </li></ol></li><li>使用校准的精度称重秤肌肉。这种快人一步使肌肉样品之间的比较分析，尤其是介入手术之后。 </li><li>放肌肉到容器中，完全用OCT化合物填充，根据肌肉的尺寸具有大约1mm的安全余量各方（ 例如 ，由铝箔制成）。 </li><li>冻结它在大约2分钟使用液氮预冷却的异戊烷。异戊烷必须开始在容器的底部，显示白色晶体。 注意:此步骤是用于保存肌肉的正确的体系结构至关重要，从而允许正确的染色和纤维分析27。 </li><li>商店样品保守OCT为在-80℃下至少24个小时。样品可以，但是，储存在-80℃数月甚至数年，如果CORRE<html> ctly冷却。 </li><li>切从肌肉的中部10μm的横截面在使用低温切片机-20℃。如果在10微米的切割是不可行的，增加的2微米的步骤厚度最多为20μm。足够尖锐切割刀片是用于此步骤是必不可少的。 </li><li>通过近似的幻灯片的横截面施加部分，以粘合剂的幻灯片。横截面会自动粘到胶粘剂幻灯片。染色前一夜保存在-20°C的幻灯片。 </li></ol> 2.染色注:可大量对肌球蛋白重链蛋白的抗体;然而，高质量的抗体可用于自动采集和分析至关重要。对于稀释和参照抗体， 见表1。 对于电子表格文件来计算正确的稀释和看需要解决量的数目，见source.jove.com/files/ftp_upload/55441/Calculator_for_muscle_fiber_typing.xlsx&quot;&gt;Supplemental文件1。 <ol><li>解冻并空气干燥该冷冻的肌肉切片用于染色过程前10分钟。 </li><li>洗涤用磷酸盐缓冲盐水（PBS）+ 0.05％的Triton X 10分钟，然后5分钟小心滑动。海卫已经显示出显著提高染色质量。有关进一步的细节讨论。 </li><li>让切片风干2分钟，使用疏水性笔（以最小化抗体的所需量），并允许额外的干燥15分钟概述在每个载玻片上单独的横截面。 </li><li>用PBS +阻止所有幻灯片0.05％的Triton X含有10％山羊血清在室温下1个小时（PBST）。 </li><li>所有稀释的初级抗体（ 表1）在PBS + 0.05％的Triton X含（PBST）10％山羊血清的鸡尾酒。应用前充分混合。曲拉通X是整个横截面的相等染色必不可少的。计算大约30微升每截面初级抗体混合物。 </li><li>对于步骤2.7-2.13确保光线足够的保护。暗淡的房间的灯和覆盖染色托盘提供足够的保护。此外，用流体填充染色托盘底部染色期间提供潮湿的环境和防止干燥。 </li><li>在室温下在潮湿染色托盘申请初级抗体（ 表1）的鸡尾酒到载玻片1个小时。 </li><li>用PBS + 0.05％的Triton X洗涤载玻片10分钟，然后用新的PBST 5分钟。 </li><li>稀释的二抗在PBS + 0.05％的Triton X（PBST）+ 10％山羊血清的鸡尾酒。计算每截面初级抗体混合物的30μL。 </li><li>应用二次抗体混合物于载玻片1个小时。 </li><li>用PBS洗涤载玻片+ 0.05％的Triton X（PBST）中10分钟，并用另外5分钟新PBST。 </li><li>施加DAPI核染色试剂盒（优选读取使用的解决方案）FOR 15 min或根据制造商的说明书进行染色细胞核。 </li><li>洗涤简要用PBS + 0.05滑动％的Triton X（PBST）中1分钟。随后，让幻灯片风干简单。 </li><li>应用荧光封固剂和盖玻片。储存于4℃下避光和24-48小时内最佳地执行成像。 </li></ol> 3.显微镜<ol><li>载片成幻灯片扫描仪。最多12个滑动取决于滑动扫描器是可行的。 </li><li>启动一个新项目。预览设置DAPI通道和2.5X透镜。对于详细的分析使用DAPI，GFP，FITC和Cy5通道和20X物镜。自动对焦是整个在DAPI通道项目的收购。 </li><li>使用每个通道下面的颜色:蓝DAPI，FITC:绿，德克萨斯红:红，cy5:黄色。 </li><li>创建使用DAPI通道每张幻灯片的预览。为了这个目的，适用于第一个标本手动对焦，并用它在整个预览分析获得每标本的预览图像。 </li><li>概述每个横截面应包括用于详细获取过程。不要画轮廓接近试样边缘，以保证所关注的整个区域的标题。 </li><li>设置曝光时间为每个通道。在大多数情况下，不同的信道的曝光参数是用于所有的横截面是相同的。 </li><li>运行自动图像采集。检查正确的收购为视图的第一个字段，以防止不正确采集的自动化过程的早期阶段。 </li><li>如果需要的话，使用桌面镜像软件建立远程控制。这允许图像采集过程的远程监视通过经由任何网络或因特网连接的载玻片扫描仪的计算机屏幕镜像到另一台计算机。虽然，不能修改对物理设置进行关于使用幻灯片，图像采集软件的虚拟环境允许监控<html>图像的正确对焦或曝光时间。此外，估计何时能完成，可以定期检查。 </li><li>图像采集完成后，通过如果纤维结构，单独的纤维和各种纤维类型是可区​​分的手动控制控制所有图像的正确自动对焦。 </li><li>重新获得正确聚焦图像领域的观点或感兴趣点面结合单一领域。对于单一领域再次取得，标志着整个项目区或图像，并用手动对焦重新获得的区域。在大多数情况下，在不同的聚焦水平的附加图像细节导致不正确聚焦的图像</li><li>对于应包括在最终的分析每个横截面，分别导出每个信道（不含DAPI）作为JPEG文件。命名并根据命名德州红，FITC和Cy5文件夹中的暴露渠道文件进行排序。 </li></ol> 4.自动纤维分析帐篷"&gt;注意:宏可以从以下网页获得:https://www.meduniwien.ac.at/hp/bionicreconstruction/macro/ <ol><li>预览每个图像使用分析前的常规图像编辑软件，并检查其染色纤维和背景之间足够的对比度。如果需要，调整对比度和亮度，以增加的差异，使用亮度和对比度调整命令。 </li><li>打开ImageJ的或斐济。使用命令打开宏" - 宏 - 插件编辑。"这显示了宏的源代码，并允许的参数快速适应。如果必要的话，对于在宏的源代码中的每个信道改变文件夹的目录。 注:对于肌纤维的分析值是基于对大鼠二头肌和蚓状肌，其它种类或肌肉可能需要不同的值。 </li><li>使用"运行"命令来启动宏。该文件夹的所有图像现在被装载到宏观，并在连续的顺序进行量化。结果是笑WN在一个新窗口。此步骤可能需要从几秒到几个小时，取决于被分析的数据量。 </li><li>导出结果窗口的电子表格文件。识别值正计数的Cy5（慢），FITC（中间）和德克萨斯红（快速）纤维和总结为总纤维数。 </li></ol>

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The authors have nothing to disclose.

<html>在这里，我们展示了一个广泛接受的方法来研究，并自动以时间有效的方式通过免疫组化定量鼠的横截面的肌纤维群。的再现性，我们提出通过步骤描述和在本研究中未描述的其它物种的应用的潜在修改的详细步骤。此外，我们还讨论了程序的优势，前提条件最佳的功能和它的局限性。 目前，许多的染色方法存在以识别肌纤维群体，并已用于肌肉纤维17...

骨骼肌组合物发生期间生理过程，如老化1，2，3的运动，4，5，6，7，或病理生理过程，例如疾病8，9，10或11的创伤深刻的变化。因此，对这些过程的结构效应研究浓缩几个字段了解的功能变化。之一的确定肌肉功能的关键方面是肌纤维的组合物。肌纤维表达不同的肌球蛋白重链（MHC）蛋白，并由此分为低，中，或快纤维7，12，13 </sup<html> &gt;，14，15，16，17。生理学上，肌肉具有不同的肌肉纤维的组合物取决于其在体内的功能。使用肌纤维打字，纤维群可以被量化，以确定适配到生理或病理生理过程7,17。在历史上，已经应用了许多耗时的方法肌纤维类型之间进行区分。为了这个目的，肌纤维或者通过在不同pH水平或肌肉酶活性肌球蛋白ATP的反应性的分类。由于不同的纤维品质不能在单个部分进行评估，要求多个横截面，以确定所有肌纤维和允许手动量化14，16，17，= "外部参照"&gt; 18，19，20，21，22。与此相反，最近的出版物中使用免疫组织化学（IHC）抗肌球蛋白重链蛋白在单个截面迅速染色多种纤维类型。基于此过程的优点，现在被认为是在肌纤维群体分析19，23，24中的黄金标准。利用改进的IHC染色方案，我们最近能够证明全自动采集整个肌肉横截面和随后的自动肌纤维定量使用开源平台的ImageJ是可行的。比手动量化，我们的程序提供在时间（手动的约10％分析）每个载玻片需要而被准确一个显著减少到±4％25 </s了&gt;。 这种方法的总体目标是使用开放源码平台来描述一种快速，可靠，方便用户独立于整个大鼠肌肉引导到自动肌纤维定量。此外，我们描述了将允许其用于其他标本，如小鼠或人的肌肉潜在的修改。

<table><tbody><tr><td>O.C.T compound</td><td>Tissue-Tek, Sakura, Netherlands</td><td></td><td>For embedding of muscle tissue</td></tr><tr><td>Isopentane</td><td></td><td></td><td>for adequate freezing of muscle tissue</td></tr><tr><td>Superfrost Ultra Plus slides</td><td>Thermo Scientific, Germany</td><td>1014356190</td><td>adhesive slides</td></tr><tr><td>phosphate buffered saline&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Triton X-100</td><td>Thermo Scientific, Germany</td><td>85112</td><td>Detergent Soluation</td></tr><tr><td>Goat serum</td><td>Thermo Scientific, Germany</td><td>50197Z</td><td>Goat Serum</td></tr><tr><td>DAKO Fluorescent Mounting Medium</td><td>Dako Denmark</td><td>S3023</td><td></td></tr><tr><td>Dako pen</td><td>Dako Denmark</td><td>S200230-2</td><td></td></tr><tr><td>TissueFAXSi plus</td><td>TissueGnostics, Vienna, Austria</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Primary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>MHC-I (Cat# BA-F8, RRID: AB_10572253)</td><td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td><td></td><td>Supernatant</td></tr><tr><td>MHC-IIa (Cat# SC-71, RRID: AB_2147165)</td><td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td><td></td><td>Supernatant</td></tr><tr><td>MHC-IIb (Cat# BF-F3, RRID: AB_2266724)</td><td>Developmental Studies Hybridoma Bank (DSHB, Iowa, USA)</td><td></td><td>Supernatant</td></tr><tr><td>&lt;strong&gt;Secondary antibodies&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Alexa&amp;nbsp;Fluor&amp;nbsp;633&amp;nbsp;Goat Anti-Mouse IgG2b&amp;nbsp;</td><td>Thermo Scientific, Germany</td><td>A-21146</td><td></td></tr><tr><td>Alexa Fluor 488 Goat Anti-Mouse IgG1&amp;nbsp;(&amp;gamma;1)</td><td>Thermo Scientific, Germany</td><td>A-21121</td><td></td></tr><tr><td>Alexa Fluor 555 Goat Anti-Mouse IgM (&amp;micro; chain)</td><td>Thermo Scientific, Germany</td><td>A-21426</td><td></td></tr><tr><td>NucBlue Fixed Cell ReadyProbes Reagent</td><td>Thermo Scientific, Germany</td><td>R37606</td><td></td></tr></tbody></table>

a rapid automated protocol for muscle fiber population analysis in rat muscle cross sections using myosin heavy chain immunohistochemistry

肌纤维群体的定量提供了更深入的了解疾病，创伤和对骨骼肌的组合物的各种其它影响的效果。各种费时的方法，传统上被用于研究纤维群体中的很多研究领域。然而，最近开发了一种基于肌球蛋白重链蛋白表达免疫组织化学方法提供了一种快速的替代在一个单一的段，以确定多种纤维类型。这里，我们提出了改进的染色品质快速，可靠和可重现的协议，允许自动采集整个横截面和纤维群体与ImageJ的自动定量。为了这个目的，嵌入式骨骼肌被切割成横截面，使用肌球蛋白重链的抗体与次级荧光抗体和DAPI对细胞核染色染色。整个横截面，然后使用滑动扫描器以获得高分辨率的复合自动扫描整个标本的照片。纤维群体分析随后以定量使用ImageJ的自动化宏慢，中间和快纤维。我们以前曾表明，该方法能够可靠地识别纤维群体的程度的±4％。此外，这种方法降低了用户间的变异性和时间每分析显著使用开源平台ImageJ的。

整个大鼠肌肉的横切片进行免疫组织化学使用来识别MHC I，IIA和IIB的肌肉纤维迅速染色。使用荧光显微镜载玻片扫描仪，整个横切片然后自动获得用于自动肌纤维分析用ImageJ的。该过程的概念是基于对于肌肉纤维的定量提供一种简单的，可靠的和时间有效的工作流程。 该过程的工作流程（ 图1）与肌肉样品的正确?...

Watch this Scientific Journal Video about A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry at JoVE.com

A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

这里，我们提出了快速肌纤维的分析，其允许改进的染色质量，并且由此自动获取和使用免费的软件ImageJ的纤维群体中量化的协议。

在使用肌球蛋白重链免疫鼠肌肉横断面的快速自动化方案为肌纤维人口分析

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle ...

a-rapid-automated-protocol-for-muscle-fiber-population-analysis-rat

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

11.2K 次观看.  Medical University of Vienna. 这里，我们提出了快速肌纤维的分析，其允许改进的染色质量，并且由此自动获取和使用免费的软件ImageJ的纤维群体中量化的协议。 

视频: A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry

Improved Protocol for Chromatin Immunoprecipitation from Mouse Skeletal Muscle

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a high content of structural proteins. Dissected limb muscles from adult mice are physically disrupted by mechanical homogenisation, or a combination of mincing and douncing, in a hypotonic buffer before formaldehyde fixation of the cell lysate. The fixed nuclei are purified by further cycles of mechanical homogenisation or douncing and sequential filtrations to remove cell debris. The purified nuclei can be sonicated immediately or at a later stage after freezing. The chromatin can be efficiently sonicated and is suitable for chromatin immunoprecipitation experiments, as illustrated by the profiles obtained for transcription factors, RNA polymerase II, and covalent histone modifications. The binding events detected using chromatin prepared by this protocol are predominantly those taking place in the muscle fiber nuclei despite the presence of chromatin from other fiber-associated satellite and endothelial cells. This protocol is therefore adapted to study gene regulation in the adult mouse skeletal muscle.

A novel protocol for the preparation of chromatin from adult mouse skeletal muscle adapted to the study of gene regulation in muscle fibers by chromatin immunoprecipitation is presented.

改进的小鼠骨骼肌染色质沉淀的协议

We describe an efficient and reproducible protocol for the preparation of chromatin from adult mouse skeletal muscle, a physically resistant tissue with a ...

科研

生物化学

Minimally Invasive Muscle Embedding (MIME) - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new muscle fibers under the right conditions. Although SCs can be harvested from muscle tissue and cultured in vitro, the resulting myoblast cells are not very effective in promoting myogenesis when transplanted into host muscle. Surgically exposing the host muscle and grafting segments of donor muscle tissue, or the isolated muscle fibers with their SCs onto host muscle, promotes better myogenesis compared to myoblast transplantation. We have developed a novel technique that we call Minimally Invasive Muscle Embedding (MIME). MIME involves passing a surgical needle through the host muscle, drawing a piece of donor muscle tissue through the needle track, and then leaving the donor tissue embedded in the host muscle so that it may act as a source of SCs for the host muscle. Here we describe in detail the steps involved in performing MIME in an immunodeficient mouse model that expresses a green fluorescent protein (GFP) in all of its cells. Immunodeficiency in the host mouse reduces the risk of immune rejection of the donor tissue, and GFP expression enables easy identification of the host muscle fibers (GFP+) and donor-cell-derived muscle fibers (GFP-). Our pilot data suggest that MIME can be used to implant an extensor digitorum longus (EDL) muscle from a donor mouse into the tibialis anterior (TA) muscle of a host mouse. Our data also suggest that when a myotoxin (barium chloride, BaCl2) is injected into the host muscle after MIME, there is evidence of donor-cell-derived myogenesis in the host muscle, with approximately 5%, 26%, 26% and 43% of the fibers in a single host TA muscle showing no host contribution, minimal host contribution, moderate host contribution, and maximal host contribution, respectively.

Minimally Invasive Muscle Embedding MIME - A Novel Experimental Technique to Facilitate Donor-Cell-Mediated Myogenesis

We describe a novel experimental technique that we call Minimally Invasive Muscle Embedding (MIME), which is based on the evidence that skeletal muscle tissue contains viable myogenic cells that can facilitate donor-cell-mediated myogenesis when implanted into a host muscle.

微创肌肉植入 (MIME)--一种促进供体细胞介导的肌的新实验技术

Skeletal muscle possesses regenerative capacity due to tissue-resident, muscle-fiber-generating (myogenic) satellite cells (SCs), which can form new ...

医学

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

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

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

小鼠骨骼肌形态及纤维型成分的半自动化分析

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

Dissection of Single Skeletal Muscle Fibers for Immunofluorescent and Morphometric Analyses of Whole-Mount Neuromuscular Junctions

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high morphological and functional plasticity. In numerous nervous system disorders, NMJ is an early pathological target resulting in neurotransmission failure, weakness, atrophy, and even in muscle fiber death. Due to its relevance, the possibility to quantitatively assess certain aspects of the relationship between NMJ components can help to understand the processes associated with its assembly/disassembly. The first obstacle when working with muscles is to gain the technical expertise to quickly identify and dissect without damaging their fibers. The second challenge is to utilize high-quality detection methods to obtain NMJ images that can be used to perform quantitative analysis. This article presents a step-by-step protocol for dissecting extensor digitorum longus and soleus muscles from rats. It also explains the use of immunofluorescence to visualize pre and postsynaptic elements of whole-mount NMJs. Results obtained demonstrate that this technique can be used to establish the microscopic anatomy of the synapsis and identify subtle changes in the status of some of its components under physiological or pathological conditions.

The ability to accurately detect neuromuscular junction components is crucial in evaluating modifications in its architecture because of pathological or developmental processes. Here we present a complete description of a straightforward method to obtain high-quality images of whole-mount neuromuscular junctions that can be used to perform quantitative measurements.

单骨骼肌肉纤维解剖，用于全山神经肌肉结的免疫荧光和形态分析

The neuromuscular junction (NMJ) is a specialized point of contact between the motor nerve and the skeletal muscle. This peripheral synapse exhibits high ...

神经科学

Characterization of Neuromuscular Junctions in Mice by Combined Confocal and Super-Resolution Microscopy

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the transmission of molecules from the nervous system to voluntary muscles, leading to contraction. They are affected in many human diseases, including inherited neuromuscular disorders such as Duchenne muscular dystrophy (DMD), congenital myasthenic syndromes (CMS), spinal muscular atrophy (SMA), and amyotrophic lateral sclerosis (ALS). Therefore, monitoring the morphology of neuromuscular junctions and their alterations in disease mouse models represents a valuable tool for pathological studies and preclinical assessment of therapeutic approaches. Here, methods for labeling and analyzing the three-dimensional (3D) morphology of the pre- and postsynaptic parts of motor endplates from murine teased muscle fibers are described. The procedures to prepare samples and measure NMJ volume, area, tortuosity and axon terminal morphology/occupancy by confocal imaging, and the distance between postsynaptic junctional folds and acetylcholine receptor (AChR) stripe width by super-resolution stimulated emission depletion (STED) microscopy are detailed. Alterations in these NMJ parameters are illustrated in mutant mice affected by SMA and CMS.

This protocol describes a method for morphometric analysis of neuromuscular junctions by combined confocal and STED microscopy that is used to quantify pathological changes in mouse models of SMA and ColQ-related CMS.

通过联合共聚焦和超分辨率显微镜表征小鼠的神经肌肉接头

Neuromuscular junctions (NMJs) are highly specialized synapses between lower motor neurons and skeletal muscle fibers that play an essential role in the ...

Visualizing the Morphological Characteristics of Neuromuscular Junction in Rat Medial Gastrocnemius Muscle

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of three essential histological components: the pre-synaptic motor axon terminals, post-synaptic nicotinic acetylcholine receptors (AchRs), and peri-synaptic Schwann cells (PSCs). In order to demonstrate the morphological characteristics of NMJ, the rat medial gastrocnemius muscle was selected as the target-tissue and examined by using multiple fluorescent staining with various kinds of biomarkers, including neurofilament 200 (NF200) and vesicular acetylcholine transporter (VAChT) for the motor nerve fibers and their pre-synaptic terminals, alpha-bungarotoxin (&#945;-BTX) for the post-synaptic nicotinic AchRs, and S100 for the PSCs. In this study, staining was performed in two groups: in the first group, samples were stained with NF200, VAChT, and &#945;-BTX, and in the second group, samples were stained with NF200, &#945;-BTX, and S100. It was shown that both protocols can effectively demonstrate the detailed structure of NMJ. Using the confocal microscope, morphological characteristics of the pre-synaptic terminals, post-synaptic receptors, and PSC were seen, and their Z-stacks images were reconstructed in a three-dimensional pattern to further analyze the spatial correlation among the different labeling. From the perspective of methodology, these protocols provide a valuable reference for investigating the morphological characteristics of NMJ under physiological conditions, which may also be suitable to evaluate the pathological alteration of NMJ, such as peripheral nerve injury and regeneration.

The protocol shows a method to examine spatial correlation among the pre-synaptic terminals, post-synaptic receptors, and peri-synaptic Schwann cells in the rat medial gastrocnemius muscle using fluorescent immunohistochemistry with different biomarkers, namely, neurofilament 200, vesicular acetylcholine transporter, alpha-bungarotoxin, and S100.

可视化大鼠内侧腓肠肌神经肌肉接头的形态学特征

The neuromuscular junction (NMJ) is a complex structure serving for the signal communication from the motor neuron to the skeletal muscle and consists of ...

Fiber Type and Subcellular-Specific Analysis of Lipid Droplet Content in Skeletal Muscle

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative prognostic factor for several other disorders such as cardiovascular disease and cancer. Excessive lipid infiltration decreases muscle mass and strength. It also results in lipotoxicity and insulin resistance depending on total intramyocellular lipid content, lipid droplet (LD) morphology, and subcellular distribution. Fiber type (oxidative vs glycolytic) is also important, since oxidative fibers have a greater capacity to utilize lipids. Because of their crucial implications in pathophysiology, in-depth studies on LD dynamics and function in a fiber type-specific manner are warranted.
Herein, a complete protocol is presented for the quantification of intramyocellular lipid content and analysis of LD morphology and subcellular distribution in a fiber type-specific manner. To this end, serial muscle cryosections were stained with the fluorescent dye Bodipy and antibodies against myosin heavy chain isoforms. This protocol enables the simultaneous processing of different muscles, saving time and avoiding possible artifacts and, thanks to a personalized macro created in Fiji, the automatization of LD analysis is also possible.

Increasing evidence indicates that excessive infiltration of lipids inside skeletal muscle results in lipotoxicity and diabetes. Here, we present a complete protocol, including tissue processing, staining with Bodipy, image acquisition, and analysis, to quantify the size, density, and subcellular distribution of lipid droplets in a fiber-type specific manner.

骨骼肌中脂质液滴含量的纤维类型和亚细胞特异性分析

Skeletal muscle lipid infiltration, known as myosteatosis, increases with obesity and ageing. Myosteatosis has also recently been discovered as a negative ...

Cryosectioning of Contiguous Regions of a Single Mouse Skeletal Muscle for Gene Expression and Histological Analyses

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene expression using adjacent regions from a single mouse skeletal muscle. Typically, it is challenging to achieve adequate homogenization of small skeletal muscle samples because buffer volumes may be too low for efficient grinding applications, yet without sufficient mechanical disruption, the dense tissue architecture of muscle limits penetration of buffer reagents, ultimately causing low RNA yield. By following the protocol reported here, 30 &#956;m sections are collected and pooled allowing cryosectioning and subsequent needle homogenization to mechanically disrupt the muscle, increasing the surface area exposed for buffer penetration. The primary limitations of the technique are that it requires a cryostat, and it is relatively low throughput. However, high-quality RNA can be obtained from small samples of pooled muscle cryosections, making this method accessible for many different skeletal muscles and other tissues. Furthermore, this technique enables matched analyses (e.g., tissue histopathology and gene expression) from adjacent regions of a single skeletal muscle so that measurements can be directly compared across applications to reduce experimental uncertainty and to reduce replicative animal experiments necessary to source a small tissue for multiple applications.

Consecutive cryo-sections are collected to enable histological applications and enrichment of RNA for gene expression measurements using adjacent regions from a single mouse skeletal muscle. High-quality RNA is obtained from 20 - 30 mg of pooled cryosections and measurements are directly compared across applications.

一个单一的鼠标骨骼肌的相邻区Cryosectioning的基因表达和组织学分析

With this method, consecutive cryosections are collected to enable both microscopy applications for tissue histology and enrichment of RNA for gene ...

生物学

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors (FAPs) and Myogenic Progenitors (MPs) in Skeletal Muscle in the Rat

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in muscle homeostasis, injury, and repair. Current protocols for FAPs identification and isolation use flow cytometry/fluorescence-activated cell sorting (FACS) and studies evaluating their function in vivo to date have been undertaken exclusively in mice. The larger inherent size of the rat allows for a more comprehensive analysis of FAPs in skeletal muscle injury models, especially in severely atrophic muscle or when investigators require substantial tissue mass to conduct multiple downstream assays. The rat additionally provides a larger selection of muscle functional assays that do not require animal sedation or sacrifice, thus minimizing morbidity and animal use by enabling serial assessments. The flow cytometry/FACS protocols optimized for mice are species specific, notably restricted by the characteristics of commercially available antibodies. They have not been optimized for separating FAPs from rat or highly fibrotic muscle. A flow cytometry/FACS protocol for the identification and isolation of FAPs and MPs from both healthy and denervated rat skeletal muscle was developed, relying on the differential expression of surface markers CD31, CD45, Sca-1, and VCAM-1. As rat-specific, flow cytometry-validated primary antibodies are severely limited, in-house conjugation of the antibody targeting Sca-1 was performed. Using this protocol, successful Sca-1 conjugation was confirmed, and flow cytometric identification of FAPs and MPs was validated by cell culture and immunostaining of FACS-isolated FAPs and MPs. Finally, we report a novel FAPs time-course in a prolonged (14 week) rat denervation model. This method provides the investigators the ability to study FAPs in a novel animal model.

Identification, Isolation, and Characterization of Fibro-Adipogenic Progenitors FAPs and Myogenic Progenitors MPs in Skeletal Muscle in the Rat

This protocol outlines a method to isolate Fibro-adipogenic progenitors (FAPs) and myogenic progenitors (MPs) from rat skeletal muscle. Utilization of the rat in muscle injury models provides increased tissue availability from atrophic muscle for the analysis and a larger repertoire of validated methods to assess muscle strength and gait in free-moving animals.

大鼠骨骼肌中纤维-脂肪源性祖细胞（FAPs）和肌源性祖细胞（MPs）的鉴定、分离和表征

Fibro-adipogenic Progenitors (FAPs) are resident interstitial cells in skeletal muscle that, together with myogenic progenitors (MPs), play a key role in ...

大鼠肌肉线粒体的共聚焦显微镜分析

Analysis of the Mitochondrial Density and Longitudinal Distribution in Rat Live-Skeletal Muscle Fibers by Confocal Microscopy

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of the mitochondrial network allows healthy mitochondria to meet cellular demands; however, the loss of this capacity has been related to the development or progression of different pathologies. In skeletal muscle, mitochondrial density and distribution changes are observed in physiological and pathological conditions such as exercise, aging, and obesity, among others. Therefore, the study of the mitochondrial network may provide a better understanding of mechanisms related to those conditions.
Here, a protocol for mitochondria imaging of live-skeletal muscle fibers from rats is described. Fibers are manually dissected in a relaxing solution and incubated with a fluorescent live-cell imaging indicator of mitochondria (tetramethylrhodamine ethyl ester, TMRE). The mitochondria signal is recorded by confocal microscopy using the XYZ scan mode to obtain confocal images of the intermyofibrillar mitochondrial (IMF) network. After that, the confocal images are processed by thresholding and binarization. The binarized confocal image accounts for the positive pixels for mitochondria, which are then counted to obtain the mitochondrial density. The mitochondrial network in skeletal muscle is characterized by a high density of IMF population, which has a periodic longitudinal distribution similar to that of T-tubules (TT).&#160;The Fast Fourier Transform (FFT) is a standard analysis technique performed to evaluate the distribution of TT that allows finding the distribution frequency and the level of their organization. In this protocol, the implementation of the FFT algorithm is described for the analysis of the longitudinal mitochondrial distribution in skeletal muscle.

作者聚焦:骨骼肌的线粒体重塑 

Here, we present a protocol to analyze changes in mitochondrial density and longitudinal distribution by live-skeletal muscle imaging using confocal microscopy for mitochondrial network scanning.

共聚焦显微镜分析大鼠活骨骼肌纤维的线粒体密度和纵向分布

The mitochondrion is an organelle that can be elongated, fragmented, and renovated according to the metabolic requirements of the cells. The remodeling of ...