Name: semi-automated analysis of mouse skeletal muscle morphology and fiber-type composition
Uploaded: 2017-08-31T13:00:00
Description: Watch this Scientific Journal Video about Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition at JoVE.com

我们感谢 Boettcher 基金会和肌萎缩侧索硬化协会 (#17 II-344) 对这项研究的支持。

所有涉及小鼠的程序均由科罗拉多大学-安舒兹医学院校园机构动物护理和使用委员会批准 (91813 (05) 1 d). 
 1. 1 天: 初级 (1 和 #176;) 免疫与牛血清白蛋白 (BSA) 阻断 <ol> <li> 小鼠后肢肌肉的空气干燥冰冻切片 (如 、胫骨前、比目鱼) 安装在带电的幻灯片上为 ~ 30 分钟 11 . 使用疏水性屏障 PAP 笔在剖面周围绘制边框. </li>
	<li> 在每张幻灯片上放置250和 #181; 5% bsa/...

<ol>
	<li>Engel, W. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essentiality+of+histo-+and+cytochemical+studies+of+skeletal+muscle+in+the+investigation+of+neuromuscular+disease.">The essentiality of histo- and cytochemical studies of skeletal muscle in the investigation of neuromuscular disease.</a> Neurol. 12, 778-784 (1962).</li><li>Dobrowolny, G., 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=Skeletal+muscle+is+a+primary+target+of+SOD1G93A-mediated+toxicity.">Skeletal muscle is a primary target of SOD1G93A-mediated toxicity.</a> Cell Metab. 8, 425-436 (2008).</li><li>Sultana, N., 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=Restricting+calcium+currents+is+required+for+correct+fiber+type+specification+in+skeletal+muscle.">Restricting calcium currents is required for correct fiber type specification in skeletal muscle.</a> Development. 143 (9), 1547-1559 (2016).</li><li>Guth, L., Samaha, F. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Procedure+for+the+histochemical+demonstration+of+actomyosin+ATPase.">Procedure for the histochemical demonstration of actomyosin ATPase.</a> Exp Neurol. 28 (2), 365-367 (1970).</li><li>Bloemberg, D., Quadrilatero, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+determination+of+myosin+heavy+chain+expression+in+rat,+mouse,+and+human+skeletal+muscle+using+multicolor+immunofluorescence+analysis.">Rapid determination of myosin heavy chain expression in rat, mouse, and human skeletal muscle using multicolor immunofluorescence analysis.</a> PLoS One. 7 (4), (2012).</li><li>Lucas, C. A., Kang, L. H., Hoh, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monospecific+antibodies+against+the+three+mammalian+fast+limb+myosin+heavy+chains.">Monospecific antibodies against the three mammalian fast limb myosin heavy chains.</a> Biochem Biophys Res Commun. 272 (1), 303-308 (2000).</li><li>Lee, C. 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=Ca2++permeation+and/or+binding+to+Cav1.1+fine-tunes+skeletal+muscle+Ca2++signaling+to+sustain+muscle+function.">Ca2+ permeation and/or binding to Cav1.1 fine-tunes skeletal muscle Ca2+ signaling to sustain muscle function.</a> Skelet Muscle. 5 (4), (2015).</li><li>Sawano, 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=A+one-step+immunostaining+method+to+visualize+rodent+muscle+fiber+type+within+a+single+specimen.">A one-step immunostaining method to visualize rodent muscle fiber type within a single specimen.</a> PLoS One. 11 (11), (2016).</li><li>Meunier, B., Picard, B., Astruc, T., Labas, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+image+analysis+tool+for+the+classification+of+muscle+fibre+type+using+immunohistochemical+staining.">Development of image analysis tool for the classification of muscle fibre type using immunohistochemical staining.</a> Histochem Cell Biol. 134 (3), 307-317 (2010).</li><li>Kammoun, M., Casser-Malek, I., Meunier, B., Picard, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simplified+immunohistochemical+classification+of+skeletal+muscle+fibres+in+mouse.">A simplified immunohistochemical classification of skeletal muscle fibres in mouse.</a> Eur J Histochem. 58 (2), 2254 (2014).</li><li>Kumar, A., Accorsi, A., Rhee, Y., Girgenrath, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Do's+and+don'ts+in+the+preparation+of+muscle+cryosections+for+histological+analysis.">Do's and don'ts in the preparation of muscle cryosections for histological analysis.</a> J Vis Exp. (99), (2015).</li><li>Schiaffino, 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=Three+myosin+heavy+chain+isoforms+in+type+2+skeletal+muscle+fibres.">Three myosin heavy chain isoforms in type 2 skeletal muscle fibres.</a> J Muscle Res Cell Motil. 10 (197), (1989).</li><li>Augusto, V., Padovani, C. R., Campos, G. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skeletal+muscle+fiber+types+in+C57Bl6j+mice.">Skeletal muscle fiber types in C57Bl6j mice.</a> Braz J Morphol Sci. 21 (2), 89-94 (2004).</li><li>Schiaffino, S., Reggiani, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiber+types+in+mammalian+skeletal+muscles.">Fiber types in mammalian skeletal muscles.</a> Physiol Rev. 91 (4), 1447-1531 (2011).</li><li>Allen, D. L., Harrison, B. C., Maass, A., Bell, M. L., Byrnes, W. C., Leinwand, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+and+skeletal+muscle+adaptations+to+voluntary+wheel+running+in+the+mouse.">Cardiac and skeletal muscle adaptations to voluntary wheel running in the mouse.</a> J Appl Physiol. 90 (5), 1900-1908 (2001).</li></ol>

在这里, 我们为骨骼肌纤维类型的鉴定提供了有用的方向。在这样的情况下, 我们描述了一种新的数据分析算法。
由于我们的结果在很大程度上证实了以前的报告5,8,10 , 并反映了我们自己的手动测量, 所以该算法看起来是准确的。尽管如此, 我们还是遇到了一些不常见的实验陷阱, 包括切片工件造成的光纤边...

众所周知, 骨骼肌是由多种类型的单纤维组成的1。最初, 两组纤维的特点是根据其收缩特性和命名, 适当, 慢抽搐 (i 型) 和快速抽搐 (II 型)。这些类别在纤维新陈代谢的基础上进一步被区别。由于 I 型纤维富含线粒体, 并依赖于氧化代谢, 它们由稳健的正烟酰胺腺嘌呤核苷酸-唑还原酶 (NADH) 酶2或琥珀酸脱氢酶 (SDH)3染色.相比之下, II 型纤维表现出较小的和可变程度的 NADH TR 酶或 SDH 染色, 分为两个快速抽搐亚组 (类型 IIA 和类型 IIB) 有些粗略的基础上, 他们的相对氧化能力。纤维的这些区别通过肌球蛋白-atp 酶染色更有效地被形象化了在 pre-incubation 在 ph 值4.0 和类型 IIB 纤维以后吸收沉淀在 ph 10.0 与类型 IIA 纤维的 I 型纤维染黑。染色中等4。
最近, 肌球蛋白重链 (MyHC) 异构体的免疫组织化学染色已经成为纤维型5的更精细的鉴别器。I 型, 类型 IIA 和类型 IIB 纤维都可以根据他们的 MyHC 轮廓精确识别。此外, 另一种快速抽搐新陈代谢-中间纤维类型, 类型 IIX, 已被确定为6。表示多个 MyHC 的混杂光纤也已被确认5,7,8。一些物种, 如猫和狒狒已知不表达类型 IIB MyHCs6。虽然 MyHC 免疫是目前最先进的评估肌肉组成, 通过这种技术获得的数据的分析是繁琐和耗时, 没有自动援助。为此, 少数的半自动方法来分析这些数据已经开发了5,9,10。在这里, 我们提出了一个相对标准的协议的肌肉纤维的免疫组织化学鉴定类型5,7,8,10,以及一个新的半自动加速分析纤维型和纤维形态的算法。

<table width="974">
	<colgroup>
		<col style="width:226pt" width="301" />
		<col style="width:140pt" width="187" />
		<col style="width:172pt" width="229" />
		<col style="width:193pt" width="257" />
	</colgroup>
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Bovine Serum Albumin</td>
			<td class="xl65" style="width:140pt" width="187">Sigma Aldrich</td>
			<td class="xl65" style="width:172pt" width="229">A9418-100G</td>
			<td class="xl66" style="width:193pt" width="257">5% in PBS</td>
		</tr>
		<tr height="41" style="height:30.75pt">
			<td class="xl65" height="41" style="width:226pt;height:30.75pt" width="301">Hydrophobic Barrier Pap Pen</td>
			<td class="xl65" style="width:140pt" width="187">Scientific Device Laboratory</td>
			<td class="xl65" style="width:172pt" width="229">9804-02</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Microscope Slides</td>
			<td class="xl65" style="width:140pt" width="187">Globe Scientific</td>
			<td class="xl65" style="width:172pt" width="229">1358W</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Coverglass</td>
			<td class="xl65" style="width:140pt" width="187">Fisher Scientific</td>
			<td class="xl65" style="width:172pt" width="229">12-544-E</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Immumount</td>
			<td class="xl65" style="width:140pt" width="187">Thermo Scientific</td>
			<td class="xl68" style="width:172pt" width="229">9990402</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Nail Polish</td>
			<td class="xl65" style="width:140pt" width="187">L&#39;Oreal</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="41" style="height:30.75pt">
			<td class="xl65" height="41" style="width:226pt;height:30.75pt" width="301">Nikon Eclipse TE-200 Inverted Fluorescence and Brightfield Microscope</td>
			<td class="xl65" style="width:140pt" width="187"></td>
			<td class="xl65" style="width:172pt" width="229">Discontinued</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">SPOT RT/KE</td>
			<td class="xl65" style="width:140pt" width="187">SPOT Imaging Solutions</td>
			<td class="xl65" style="width:172pt" width="229">RT940</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Dell Optiplex</td>
			<td class="xl65" style="width:140pt" width="187"></td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl66"></td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">BA-F8 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monoclonal mouse IgG2b; 1:50</td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">SC-71 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monclonal mouse IgG1; 1:50</td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">BF-F3 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monoclonal mouse IgGM; 1:50</td>
		</tr>
		<tr height="61" style="height:45.75pt">
			<td class="xl65" height="61" style="width:226pt;height:45.75pt" width="301">6H1 Primary Antibody</td>
			<td class="xl65" style="width:140pt" width="187">Developmental Studies Hybridoma Bank at the University of Iowa</td>
			<td class="xl65" style="width:172pt" width="229"></td>
			<td class="xl70">monoclonal mouse IgGM; 1:50</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 594 anti-IgG2b</td>
			<td class="xl65" style="width:140pt" width="187">Invitrogen</td>
			<td class="xl65" style="width:172pt" width="229">A21145</td>
			<td class="xl66">goat anti-mouse; 1:200</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 488 anti-IgG1</td>
			<td class="xl65" style="width:140pt" width="187">Invitrogen</td>
			<td class="xl65" style="width:172pt" width="229">A21121</td>
			<td class="xl66">goat anti-mouse;1:200</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Alexa Fluor 594 anti-IgGM</td>
			<td class="xl65" style="width:140pt" width="187">Invitrogen</td>
			<td class="xl65" style="width:172pt" width="229">A21044</td>
			<td class="xl66">goat anti-mouse;1:200</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">OCT</td>
			<td class="xl65" style="width:140pt" width="187">Sakura Finetek</td>
			<td class="xl68" style="width:172pt" width="229">4583</td>
			<td class="xl66"></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">isopentane</td>
			<td class="xl65" style="width:140pt" width="187">Fisher Scientific</td>
			<td class="xl65" style="width:172pt" width="229">O3551-4</td>
			<td class="xl66">cool with liguid nitrogen</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">PBS</td>
			<td class="xl65" style="width:140pt" width="187">Fisher Bioreagents</td>
			<td class="xl65" style="width:172pt" width="229">BP665-1</td>
			<td class="xl66">10X, dilute to 1X</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Kim wipes</td>
			<td class="xl66">Kimberly-Clark</td>
			<td class="xl65" style="width:172pt" width="229">06-666A</td>
			<td class="xl66"></td>
		</tr>
	</tbody>
</table>

semi-automated analysis of mouse skeletal muscle morphology and fiber-type composition

近年来, 肌球蛋白-atp 酶染色法对骨骼肌纤维类型的差异进行了最佳的可视化。近年来, 肌球蛋白重链 (MyHC) 异构体的免疫组织化学染色逐渐成为纤维型的一种较细的鉴别。I 型, 类型 IIA, 类型 IIX 和类型 IIB 纤维现在可以根据他们的 MyHC 轮廓精确识别;但是, 手动分析这些数据可能是缓慢和向下-右单调乏味的。在这方面, 快速、准确地评估纤维类成分和形态是一个非常理想的工具。在这里, 我们提出了一项协议的最先进的免疫组化染色的 MyHCs 在冰冻切片从小鼠后肢肌肉与一个新的半自动算法, 加速分析纤维型和纤维形态。正如预期的, 比目鱼肌显示染色 I 型和类型 IIA 纤维, 但不是类型 IIX 或类型 IIB 纤维。另一方面, 胫骨前肌主要由 IIX 型和 IIB 纤维组成, 一小部分的 IIA 型纤维和很少或没有 I 型纤维。为了测量纤维形态的不同方面 (即、横截面积 (CSA)、最大和最小 Feret 直径), 采用了几种图像变换来生成概率图。然后将这些参数获得的值与手动获取的值进行比较。在 CSA、最大或最小 Feret 直径 (全部为p和 #62; 0.05) 中, 两种分析方法均无显著性差异, 说明了本法的准确性。因此, 我们的免疫分析协议可以应用于对许多衰老和肌病模型中肌肉组成的影响的研究。

后肢肌肉 (即, 胫骨前, 比目鱼) 解剖从一个未知年龄的雄性 C57BL/6 小鼠的闪光冻结了一个塑料模具中含有的肌肉在 OCT 化合物在液氮冷却戊。然后, 使用 cryotome, 8-10 µm 串行部分被切割在-20 ° c 和转移到不同的正电荷玻璃幻灯片12。
我们选择胫骨前和比目鱼, 因为这些肌肉主要由快速和慢抽搐纤维组成, 分...

Watch this Scientific Journal Video about Semi-automated Analysis of Mouse Skeletal Muscle Morphology and Fiber-type Composition at JoVE.com

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

肌球蛋白重链异构体的免疫组织化学染色已成为骨骼肌纤维型 (即、i 型、类型 IIA、类型 IIX、类型 IIB) 的最先进的鉴别器。在这里, 我们提出了一个染色协议和一个新的半自动算法, 促进纤维型和纤维形态的快速评估。

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 ...

Research

JoVE Journal

Medicine

Myo-mechanical Analysis of Isolated Skeletal Muscle

离体骨骼肌肌的力学分析

To assess the in vivo effects of therapeutic interventions for the treatment of muscle disease 1,2,3, quantitative methods are needed that measure force ...

科研

医学

Skeletal Muscle Gender Dimorphism from Proteomics

骨骼肌蛋白质组学性别异形

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably ...

Human Skeletal Muscle Biopsy Procedures Using the Modified Bergstr&#246;m Technique

Human Skeletal Muscle Biopsy Procedures Using the Modified BergstrÃƒÆ’Ã‚Â¶m Technique

人骨骼肌活检手术使用修改伯格斯坦技术

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly ...

Quantitative Magnetic Resonance Imaging of Skeletal Muscle Disease

骨骼肌疾病的定量磁共振成像

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of ...

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

Human Vastus Lateralis Skeletal Muscle Biopsy Using the Weil-Blakesley Conchotome

人的<em&gt;股外侧肌</em&gt;骨骼肌活检使用韦尔 - Blakesley Conchotome

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and ...

Induction and Assessment of Exertional Skeletal Muscle Damage in Humans

诱导和人类劳力骨骼肌损伤评估

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery ...

Echocardiographic and Histological Examination of Cardiac Morphology in the Mouse

超声心动图与小鼠心脏形态学的组织学检查

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed ...

Body Composition and Metabolic Caging Analysis in High Fat Fed Mice

高脂喂养小鼠体内成分及代谢锁定分析

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate ...

Quantitative Analysis of Cellular Composition in Advanced Atherosclerotic Lesions of Smooth Muscle Cell Lineage-Tracing Mice

平滑肌细胞近系追踪小鼠晚期动脉粥样硬化病变细胞成分的定量分析

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel ...

Segmentation and Linear Measurement for Body Composition Analysis using Slice-O-Matic and Horos

使用切片-O-马蒂奇和霍罗斯进行身体成分分析的细分和线性测量

Body composition is associated with risk of disease progression and treatment complications in a variety of conditions. Therefore, quantification of ...