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
1CD Laboratory for the Restoration of Extremity Function, Division of Plastic and Reconstructive Surgery, Department of Surgery,Medical University of Vienna, 2Core Facility Imaging, Core Facilities,Medical University Vienna, 3Department of Hand, Plastic and Reconstructive Surgery, Burn Center, BG Trauma Center Ludwigshafen, Plastic and Hand Surgery,University of Heidelberg
Summary
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。
Abstract
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。
Introduction
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 >，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”> 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>。
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。
Protocol
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。 <li…
Representative Results
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?…
Discussion
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…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
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。
Materials
| O.C.T compound | Tissue-Tek, Sakura, Netherlands | For embedding of muscle tissue | |
| Isopentane | for adequate freezing of muscle tissue | ||
| Superfrost Ultra Plus slides | Thermo Scientific, Germany | 1014356190 | adhesive slides |
| phosphate buffered saline | |||
| Triton X-100 | Thermo Scientific, Germany | 85112 | Detergent Soluation |
| Goat serum | Thermo Scientific, Germany | 50197Z | Goat Serum |
| DAKO Fluorescent Mounting Medium | Dako Denmark | S3023 | |
| Dako pen | Dako Denmark | S200230-2 | |
| TissueFAXSi plus | TissueGnostics, Vienna, Austria | ||
| Primary antibodies | |||
| MHC-I (Cat# BA-F8, RRID: AB_10572253) | Developmental Studies Hybridoma Bank (DSHB, Iowa, USA) | Supernatant | |
| MHC-IIa (Cat# SC-71, RRID: AB_2147165) | Developmental Studies Hybridoma Bank (DSHB, Iowa, USA) | Supernatant | |
| MHC-IIb (Cat# BF-F3, RRID: AB_2266724) | Developmental Studies Hybridoma Bank (DSHB, Iowa, USA) | Supernatant | |
| Secondary antibodies | |||
| Alexa Fluor 633 Goat Anti-Mouse IgG2b | Thermo Scientific, Germany | A-21146 | |
| Alexa Fluor 488 Goat Anti-Mouse IgG1 (γ1) | Thermo Scientific, Germany | A-21121 | |
| Alexa Fluor 555 Goat Anti-Mouse IgM (µ chain), | Thermo Scientific, Germany | A-21426 | |
| NucBlue Fixed Cell ReadyProbes Reagent | Thermo Scientific, Germany | R37606 |
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Citazione di questo articolo
Bergmeister, K. D., Gröger, M., Aman, M., Willensdorfer, A., Manzano-Szalai, K., Salminger, S., Aszmann, O. C. A Rapid Automated Protocol for Muscle Fiber Population Analysis in Rat Muscle Cross Sections Using Myosin Heavy Chain Immunohistochemistry. J. Vis. Exp. (121), e55441, doi:10.3791/55441 (2017).