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/磷酸盐缓冲盐水 (bsa/PBS), 以阻止非特异性抗体结合。在室温下孵育1小时的幻灯片 </li>
	<li> 在阻塞时, 准备1:50 稀释1和 #176; 抗体上清 5% BSA/PBS (均为小鼠单克隆抗体; 请参阅 表 1 6 , 12 ); 准备250和 #181;每张幻灯片的解决方案。保持1和 #176; 抗体储存和稀释在冰上. </li>
</ol> <img alt="Table 1" src="/files/ftp_upload/56024/56024table1.jpg"/> 表 1: 用于区分 MyHCs 的主要抗体. 
<ol start="4"> <li> 跟随 5% BSA/PBS 拦截解决方案的愿望, 添加250和 #181; L 1 和 #176; 抗体稀释到适当的幻灯片。添加250和 #181; L 5% BSA/PBS 到次要 (2 和 #176;) 抗体仅阴性控制幻灯片. </li>
	<li> 在潮湿环境中孵育4和 #176; C 为 24-48 h. 
	注: 用带有湿滤纸的铝箔盖的培养皿可以作为合适的孵化室. </li>
</ol> 2。2天: 2 和 #176; 免疫与荧光抗体 <ol> <li> 吸气1和 #176; 抗体溶液或控制 5% BSA/PBS 溶液。冲洗所有幻灯片三次, 10 分钟, 5% BSA. </li>
	<li> 洗涤时, 准备1:200 稀释的纯化, 荧光共轭2和 #176; 抗体在 5% BSA/PBS (见 表 2 , 所有都是山羊 anti-mouse)。为每张幻灯片准备250和 #181;保持2和 #176; 在黑暗中荧光共轭抗体在冰上. </li>
</ol> <img alt="Table 2" src="/files/ftp_upload/56024/56024table2.jpg"/> 表 2: 用于可视化 MyHCs 的初级抗体识别的荧光共轭二级抗体. 
<ol start="3"> <li> 以下是 5% BSA/PBS 阻断解决方案的第三个应用的愿望, 添加250和 #181; L 2 和 #176; 所有幻灯片的抗体稀释。在阴暗潮湿的环境中, 室温下孵育90分钟. </li>
	<li> 吸气2和 #176; 抗体溶液。冲洗所有幻灯片三次, 10 分钟, 5% BSA/PBS. </li>
	<li> 用 PBS 冲洗。干燥10分钟. </li>
	<li> 安装 coverglass 具有非永久性、低的水性安装介质. </li>
	<li> 干燥时, 用指甲油密封滑动边。干燥和储存在一个黑暗的 slidebox. </li>
</ol> 3。带荧光显微镜的成像幻灯片 <ol> <li> 用一个小的、70% 乙醇浸泡过的实验室擦拭擦除幻灯片. </li>
	<li> 使用装有照相设备的荧光显微镜获取 immunostained 肌肉部分的数字图像 (请参阅 材料表 )。
	<ol> <li> 选择大约 1 mm 2 (10X 目标, 1.4 NA) 的区域. </li>
		<li> 查看 Alexa 488-共轭2和 #176; 抗体荧光通过 505 nm long-pass 过滤器。查看由 594-共轭2和 #176 的激励产生的荧光; 通过 595 nm long-pass 过滤器的抗体。用装有兼容成像软件的 PC 计算机将图像数字化。将缩放栏包括在图像软件中以供以后分析. </li>
	</ol> </li> </ol> 4。与斐济的图像分析 注意: 在进行分析之前, 必须通过 https://imagej.net/Fiji/Downloads 安装斐济 (从国立卫生研究院免费提供)。此外, 在 补充文件 中提供了此过程中使用的宏。将宏文件放在易于访问的目录中. 
<ol> <li> 在斐济加载选定节的明图像。导航到插件和 #8594; 分割和 #160; #8594; 训练 Weka 切分. </li>
	<li> 为了规定部分的蜂窝区域, 在光纤上绘制一条线, 然后单击并 #34; 添加到类1和 #34;。然后, 在纤维之间的空间上画一条线, 然后点击和 #34; 添加到类2和 #34; 标记细胞外领域。重复, 直到每个类中有5-10 标签。
	<ol> <li> 单击 "训练分类器"。在生成的红色和绿色叠加上, 如有必要, 手动添加更多标签 (请参见步骤 4.2) 以不正确地分割图像的片段。重复直到纤维 (红色) 与纤维 (绿色) 之间的空间适当分离. </li>
	</ol> </li> <li> 单击并 #34; 获取概率和 #34; 并将图像保存在新标记的文件夹中 </li>
	<li> 重复上述过程 (步骤 4.2-4.3), 并从同一字段中取出荧光图像。标签 immunostained 纤维为和 #34; 类1和 #34; 和所有荧光地区作为和 #34; 类2和 #34;。将生成的概率映射保存到存储已处理明映像的同一文件夹中. </li>
	<li> 在斐济打开以前保存的概率映射之一。在成像软件提供的刻度线上绘制一条直线。去分析和 #160; #8594; 设置比例。将参数 ( 即 、已知距离、长度单位) 更改为缩放栏给定的相应值, 然后检查和 #34; 全局和 #34; 框以标准化每个图像的比例. </li>
	<li> 导航到插件和 #160; #8594; 宏和 #160; #8594; 运行和 #160; #8594; 泰亚吉 等 al. 光纤量化宏. ijm (请参见 补充文件 )。随即会出现一个导航窗格。打开图像和 #39 的主机文件夹;将出现一个对话框。对于每个对话框, 更改和 #34; 背景和 #34; 下拉列表值和 #34; 光. #34; 注意: 该文件夹现在将填充图像的光纤轮廓和电子表格软件文件的结果 (CSA 和 Feret 直径是最相关的量化纤维形态;测量参数可以在分析集合测量中进行调整)。斐济的孔隙分析功能产生的纤维形态数据将自动转移到电子表格软件文件中, 用于荧光和亮场图像. </li>
	<li> 通过将字段中的荧光纤维数量除以相应的明亮场图像中的总光纤数来计算字段中表示给定 MyHC 的纤维的分数. </li>
</ol>

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<li>Lee, C. S., et al. <a target="_blank" href="https://skeletalmusclejournal.biomedcentral.com/articles/10.1186/s13395-014-0027-1">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/pubmed?term=((A+one-step+immunostaining+method+to+visualize+rodent+muscle+fiber+type+within+a+single+specimen%5BTitle%5D)+AND+%22PLoS+One%22%5BJournal%5D)">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/pubmed?term=((Development+of+image+analysis+tool+for+the+classification+of+muscle+fibre+type+using+immunohistochemical+staining%5BTitle%5D)+AND+%22Histochem+Cell+Biol%22%5BJournal%5D)">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/pubmed?term=((A+simplified+immunohistochemical+classification+of+skeletal+muscle+fibres+in+mouse%5BTitle%5D)+AND+%22Eur+J+Histochem%22%5BJournal%5D)">A simplified immunohistochemical classification of skeletal muscle fibres in mouse.</a> Eur J Histochem. 58 (2), 2254(2014).</li>
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<li>Schiaffino, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Three+myosin+heavy+chain+isoforms+in+type+2+skeletal+muscle+fibres%5BTitle%5D)+AND+%22J+Muscle+Res+Cell+Motil%22%5BJournal%5D)">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="https://www.researchgate.net/publication/228866300_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>
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作者没有利益冲突的披露。

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><tbody><tr><td>Bovine Serum Albumin</td><td>Sigma Aldrich</td><td>A9418-100G</td><td>5% in PBS</td></tr><tr><td>Hydrophobic Barrier Pap Pen</td><td>Scientific Device Laboratory</td><td>9804-02</td><td></td></tr><tr><td>Microscope Slides</td><td>Globe Scientific</td><td>1358W</td><td></td></tr><tr><td>Coverglass</td><td>Fisher Scientific</td><td>12-544-E</td><td></td></tr><tr><td>Immumount</td><td>Thermo Scientific</td><td>9990402</td><td></td></tr><tr><td>Nail Polish</td><td>L&#039;Oreal</td><td></td><td></td></tr><tr><td>Nikon Eclipse TE-200 Inverted Fluorescence and Brightfield Microscope</td><td></td><td>Discontinued</td><td></td></tr><tr><td>SPOT RT/KE</td><td>SPOT Imaging Solutions</td><td>RT940</td><td></td></tr><tr><td>Dell Optiplex</td><td></td><td></td><td></td></tr><tr><td>BA-F8 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monoclonal mouse IgG2b; 1:50</td></tr><tr><td>SC-71 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monclonal mouse IgG1; 1:50</td></tr><tr><td>BF-F3 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monoclonal mouse IgGM; 1:50</td></tr><tr><td>6H1 Primary Antibody</td><td>Developmental Studies Hybridoma Bank at the University of Iowa</td><td></td><td>monoclonal mouse IgGM; 1:50</td></tr><tr><td>Alexa Fluor 594 anti-IgG2b</td><td>Invitrogen</td><td>A21145</td><td>goat anti-mouse; 1:200</td></tr><tr><td>Alexa Fluor 488 anti-IgG1</td><td>Invitrogen</td><td>A21121</td><td>goat anti-mouse;1:200</td></tr><tr><td>Alexa Fluor 594 anti-IgGM</td><td>Invitrogen</td><td>A21044</td><td>goat anti-mouse;1:200</td></tr><tr><td>OCT</td><td>Sakura Finetek</td><td>4583</td><td></td></tr><tr><td>isopentane</td><td>Fisher Scientific</td><td>O3551-4</td><td>cool with liguid nitrogen</td></tr><tr><td>PBS</td><td>Fisher Bioreagents</td><td>BP665-1</td><td>10x, dilute to 1x</td></tr><tr><td>Kim wipes</td><td>Kimberly-Clark</td><td>06-666A</td><td></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 ...

semi-automated-analysis-mouse-skeletal-muscle-morphology-fiber-type

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10.4K 次观看.  University of Colorado School of Medicine. 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。

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

Myo-mechanical Analysis of Isolated Skeletal Muscle

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 generation and fatigability in treated muscle. We describe a detailed approach to evaluating myo-mechanical properties in freshly explanted hindlimb muscle from the mouse. We describe the atraumatic harvest of mouse extensor digitorum longus muscle, mounting the muscle in a muscle strip myograph (Model 820MS; Danish Myo Technology), and the measurement of maximal twitch and tetanic tension, contraction time, and half-relaxation time, using a square pulse stimulator (Model S48; Grass Technologies). Using these measurements, we demonstrate the calculation of specific twitch and tetanic tension normalized to muscle cross-sectional area, the twitch-to-tetanic tension ratio, the force-frequency relationship curve and the low frequency fatigue curve 4. This analysis provides a method for quantitative comparison between therapeutic interventions in mouse models of muscle disease 1,2,3,5, as well as comparison of the effects of genetic modification on muscle function 6,7,8,9.

To assess the in vivo effects of therapeutic interventions for muscle disease, methods are needed to quantitate force generation and fatigability in treated muscle. We detail an approach to evaluating myo-mechanical properties in explanted mouse hindlimb muscle. This analysis provides a robust approach to quantitating the effects of genetic modification on muscle function, as well as comparison of therapies in mouse models of muscle disease.

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

Gross contraction in skeletal muscle is primarily determined by a relatively small number of contractile proteins, however this tissue is also remarkably adaptable to environmental factors1 such as hypertrophy by resistance exercise and atrophy by disuse. It thereby exhibits remodeling and adaptations to stressors (heat, ischemia, heavy metals, etc.)2,3. Damage can occur to muscle by a muscle exerting force while lengthening, the so-called eccentric contraction4. The contractile proteins can be damaged in such exertions and need to be repaired, degraded and/or resynthesized; these functions are not part of the contractile proteins, but of other much less abundant proteins in the cell. To determine what subset of proteins is involved in the amelioration of this type of damage, a global proteome must be established prior to exercise5 and then followed subsequent to the exercise to determine the differential protein expression and thereby highlight candidate proteins in the adaptations to damage and its repair. Furthermore, most studies of skeletal muscle have been conducted on the male of the species and hence may not be representative of female muscle. 
In this article we present a method for extracting proteins reproducibly from male and female muscles, and separating them by two-dimensional gel electrophoresis followed by high resolution digital imaging6. This provides a protocol for spots (and subsequently identified proteins) that show a statistically significant (p &lt; 0.05) two-fold increase or decrease, appear or disappear from the control state. These are then excised, digested with trypsin and separated by high-pressure liquid chromatography coupled to a mass spectrometer (LC/MS) for protein identification (LC/MS/MS)5. This methodology (Figure 1) can be used on many tissues with little to no modification (liver, brain, heart etc.).

A straight-forward set of methods to isolate and determine the identity of the most abundant proteins expressed in skeletal muscle. About 800 spots are discerned on a two-dimensional gel from 10 mg muscle; this allows for the determination of gender-specific protein expression. These methods will give equivalent results in most tissues.

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 ...

Assessment of Right Ventricular Structure and Function in Mouse Model of Pulmonary Artery Constriction by Transthoracic Echocardiography

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, the RV is significantly affected in pulmonary diseases such as pulmonary artery hypertension (PAH). In addition, the RV is remarkably sensitive to cardiac pathologies, including left ventricular (LV) dysfunction, valvular disease or RV infarction4. To understand the role of RV in the pathogenesis of cardiac diseases, a reliable and noninvasive method to access the RV structurally and functionally is essential.
A noninvasive trans-thoracic echocardiography (TTE) based methodology was established and validated for monitoring dynamic changes in RV structure and function in adult mice. To impose RV stress, we employed a surgical model of pulmonary artery constriction (PAC) and measured the RV response over a 7-day period using a high-frequency ultrasound microimaging system. Sham operated mice were used as controls. Images were acquired in lightly anesthetized mice at baseline (before surgery), day 0 (immediately post-surgery), day 3, and day 7 (post-surgery). Data was analyzed offline using software.
Several acoustic windows (B, M, and Color Doppler modes), which can be consistently obtained in mice, allowed for reliable and reproducible measurement of RV structure (including RV wall thickness, end-diastolic&#160;and end-systolic dimensions), and function (fractional area change, fractional shortening, PA peak velocity, and peak pressure gradient) in normal mice and following PAC.
Using this method, the pressure-gradient resulting from PAC was accurately measured in real-time using Color Doppler mode and was comparable to direct pressure measurements performed with a Millar high-fidelity microtip catheter. Taken together, these data demonstrate that RV measurements obtained from various complimentary views using echocardiography are reliable, reproducible and can provide insights regarding RV structure and function. This method will enable a better understanding of the role of RV cardiac dysfunction.

Right ventricle (RV) dysfunction is critical to the pathogenesis of cardiovascular disease, yet limited methodologies are available for its evaluation. Recent advances in ultrasound imaging provide a noninvasive and accurate option for longitudinal RV study. Herein, we detail a step-by-step echocardiographic method using a murine model of RV pressure overload.

右心室结构和功能的肺动脉缩窄经胸超声小鼠模型的评估

Emerging clinical data support the notion that RV dysfunction is critical to the pathogenesis of cardiovascular disease and heart failure1-3. Moreover, ...

MyDoBID- 一种有效识别肌肉纤维类型的新技术

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

作者聚焦:使用 MyDoBID 技术破译骨骼肌纤维类型的奥秘 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

人体骨骼肌的纤维类型鉴定

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

生物化学

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

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 ...

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

Quantification of muscle fiber populations provides a deeper insight into the effects of disease, trauma, and various other influences on skeletal muscle composition. Various time-consuming methods have traditionally been used to study fiber populations in many fields of research. However, recently developed immunohistochemical methods based on myosin heavy chain protein expression provide a quick alternative to identify multiple fiber types in a single section. Here, we present a rapid, reliable and reproducible protocol for improved staining quality, allowing automatic acquisition of whole cross sections and automatic quantification of fiber populations with ImageJ. For this purpose, embedded skeletal muscles are cut in cross sections, stained using myosin heavy chains antibodies with secondary fluorescent antibodies and DAPI for cell nuclei staining. Whole cross sections are then scanned automatically using a slide scanner to obtain high-resolution composite pictures of the entire specimen. Fiber population analyses are subsequently performed to quantify slow, intermediate and fast fibers using an automated macro for ImageJ. We have previously shown that this method can identify fiber populations reliably to a degree of &#177;4%. In addition, this method reduces inter-user variability and time per analyses significantly using the open source platform ImageJ.

Here, we present a protocol for rapid muscle fiber analyses, which allows improved staining quality, and thereby automatic acquisition and quantification of fiber populations using the freely available software ImageJ.

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 ...

生物学

High-Throughput Contractile Measurements of Hydrogel-Embedded Intact Mouse Muscle Fibers Using an Optics-Based System

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches involve either differentiating myogenic progenitor cells into immature myotubes or the short-term ex vivo culture of isolated individual muscle fibers. A key benefit of ex vivo culture over in vitro is the retention of the complex cellular architecture and contractile characteristics. Here, we detail an experimental protocol for the isolation of intact flexor digitorum brevis muscle fibers from mice and their subsequent ex vivo culture. In this protocol, muscle fibers are embedded in a fibrin-based and basement membrane matrix hydrogel to immobilize the fibers and maintain their contractile function. We then describe methods to assess the muscle fiber contractile function using an optics-based, high-throughput contractility system. The embedded muscle fibers are electrically stimulated to induce contractions, after which their functional properties, such as sarcomere shortening and contractile velocity, are assessed using optics-based quantification. Coupling muscle fiber culture with this&#160;system allows for high-throughput testing of the effects of pharmacological agents on contractile function and ex vivo studies of genetic muscle disorders. Finally, this protocol can also be adapted to study dynamic cellular processes in muscle fibers using live-cell microscopy.

Skeletal muscle function can be assessed by quantifying the contractility of isolated muscle fibers, traditionally using laborious, low-throughput approaches. Here, we describe an optics-based, high-throughput method to quantify the contractility of hydrogel-embedded muscle fibers. This approach has applications for drug screening and therapeutic development.

使用基于光学的系统对水凝胶嵌入的完整小鼠肌纤维进行高通量收缩测量

In vitro cell culture is a powerful tool to assess cellular processes and test therapeutic strategies. For skeletal muscle, the most common approaches ...

小鼠骨骼肌形态及纤维型成分的半自动化分析

摘要

探索更多视频

离体骨骼肌肌的力学分析

骨骼肌蛋白质组学性别异形

右心室结构和功能的肺动脉缩窄经胸超声小鼠模型的评估

人体骨骼肌的纤维类型鉴定

人体骨骼肌的纤维类型鉴定

人体骨骼肌的纤维类型鉴定

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

骨骼肌中脂质液滴含量的纤维类型和亚细胞特异性分析

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

使用基于光学的系统对水凝胶嵌入的完整小鼠肌纤维进行高通量收缩测量