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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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界不清晰。在分析之前, 通过仔细剖分和对处理过的部分进行警惕检查, 可以最大限度地减少这些工件的影响。特别是, 不太理想的图像分割可以通过在同一图像上的多重更改来规避, 以达到更鲜明的对比度。
我们的协议中最关键的步骤是步骤 4.2, 我们通过在给定的纤维上画一条线来规定该部分的细胞和胞外区域, 从而区分 "1 类" 或 "2 类" 区域。显然, 这一手动区分是至关重要的, 因为它决定了肌肉纤维的边界;此步骤中的错误将对光纤尺寸的评估产生负面影响。
在这项研究中, 我们在一个给定的序列部分中只探测一个 MyHC 的部分。然而, 一些纤维, 如混杂纤维, 表达两种类型的 MyHC, 表明重叠的新陈代谢/功能配置文件 (例如, 类型 IIX 和类型 IIB)。因此, 纤维类型的分数总结为一个值和 #62; 1 的比目鱼和胫骨前 (图 4a)。混合纤维可以被识别为双, 甚至三重, 标签协议, 如那些由李et al.分别为7和 Bloemberg 和 Quadrilatero5。重要的是, 我们的分析方法可以应用于这些更复杂的染色协议。我们的协议的另一个限制是它不是完全自动的;然而, 我们认为, 这一小的让步, 确保我们的方法不妥协的速度, 以严格的僵硬。
在本研究中观察到的比目鱼肌和胫骨前肌肉的 MyHC 成分反映了先前研究的结果5,6,7,10,14, 与先前关于 CSA 评估的研究有一些对比。例如, 我们对平均 CSA 的测量比 Bloemberg 和 Quadrilatero5和艾伦et al所报告的都要小一些。15特别是, 我们的 CSA 测量范围从539.86 ±24.44 µm2到1319.50 ±57.47 µm2跨越光纤类型, 而它们的跨度约735.7 ±31.2 µm2到3073.8 ±51.3 µm2。然而, 我们的价值观更像那些被观察到的et al.13和卢卡斯et al.6谁报告的平均 csa 范围从436±605µm2到2404±412.5 µm2和815±221µm2到1904±570µm2分别。因此, 在整个文献中存在着小鼠后肢纤维形态分析的一些变异性。
免疫组织化学提供了一种有效的方法, 标签和分类肌肉纤维的基础上, 他们的 MyHC 内容。准确定量化免疫组织化学实验是对数据做出有意义的结论的关键;为此, 一个可以自动化繁琐的分析部分的方法是有用的, 特别是当数百甚至数千的纤维需要被测量和分类。除了提供类似于手动观察的测量, 我们的算法还提供了对这些数据的精确评估, 而不带有偏差;这比手工测量的优势, 确保科学的严谨。在这方面, 我们的方法在描述影响肌肉完整性的基因和实验操作的影响方面可能是有用的。具体地说, MyHC 免疫组织化学结合我们的快速分析方法可以用来评估肌肉的形态学和代谢变化的疾病模型, 如肌肉营养, ALS 和癌症恶病质。此外, 还可评估预防萎缩或引起肥大的干预措施的功效。

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。

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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Bovine Serum Albumin</td>
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			<td class="xl65" style="width:172pt" width="229">A9418-100G</td>
			<td class="xl66" style="width:193pt" width="257">5% in PBS</td>
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			<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>
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			<td class="xl65" height="21" style="width:226pt;height:15.75pt" width="301">Microscope Slides</td>
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			<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>
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		<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>
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			<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, 分别为13,14。在免疫组织化学染色的个人 I 型, IIA, IIX 和 IIB MyHCs, 明和荧光图像捕获。图 1的左面板显示了在表 1中列出的 immunostained 与 I 型、IIA、IIX 和 IIB MyHC 特定抗体的比目鱼肌的部分。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56024/56024fig1.jpg"> 
图 1: MyHC I 型、IIA、IIX 和 IIB 在小鼠比目鱼肌中的免疫组织化学染色.左面板显示从一只小鼠比目鱼肌的部分获得的荧光图像, 其主要抗体针对 I 型 (BA-F8;A), 类型 IIA (SC-71;B), 键入 IIX (6H1;C) 和类型 IIB (BF-F3;D) MyHCs。右面板显示了在相应的左面板中所描述的实验中使用的主抗体被省略的部分。条形 = 100 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56024/56024fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
具体来说, 我们观察了大量的纤维表示类型 I (图 1A, 左) 和类型 IIA MyHCs (图 1B, 左) 与相当数量的剩余纤维显示阳性染色的类型IIX MyHCs (图 1C, 左侧)。相比之下, 实际上没有任何类型的 IIB 光纤 (图 1D, 左侧)。这些观察与先前的报告一致, 比目鱼肌主要由 I 型、型 IIA 和型 IIX 纤维组成 (较少), 几乎没有类型的 IIB 纤维5,10,12。重要的是, 没有观察到的部分, 其中的主要抗体已被省略的协议, 证明特异性抗体 (图 1A-D, 右面板)。
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56024/56024fig2.jpg"> 
图 2: MyHC 型 IIB、IIX、IIA 和 I 在小鼠胫骨前肌的免疫组织化学染色.左面板显示从小鼠胫骨前肌的部分获得的荧光图像, 其主要目的是对类型 IIB (a)、类型 IIX (B)、类型 IIA (C) 和 I 类型 (D) MyHCs 进行探测。右面板显示了在相邻左面板中所描述的实验中使用的主抗体被省略的部分。条形 = 100 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56024/56024fig2large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
我们还用上述抗体染色胫骨前肌。胫骨前肌肉主要由类型 IIB 和类型 IIX MyHC 阳性纤维 (图 2A-b, 左面板) 组成, 从纤维表达型 IIA MyHCs (图 2C, 左) 的贡献较小;几乎没有观察到表达 I 型 MyHC 的纤维 (图 2D, 左侧)。我们的数据表明, 小鼠胫骨前主要由快速抽搐型 IIA 组成, 类型 IIB 和类型 IIX 纤维是一致的早期研究5,10,13。同样, 在从协议中省略主抗体的部分 (图 2A-D, 右面板) 中没有观察到任何染色。
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56024/56024fig3.jpg"> 
图 3: 迭代计算机化光纤分段.明图像的鼠标胫骨前段 (a)。使用我们的半自动算法 (B) 对同一图像进行转换。显示免疫型 IIA MyHC (C) 的同一剖面的荧光图像。仅显示类型 IIA MyHC 表示纤维 (D) 的转换图像。条形 = 100 µm.<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56024/56024fig3large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
为了进行形态分析, 还得到了 immunostained 场的明图像。图 3a中显示的示例来自胫骨前肌肉部分, 该 SC-71 抗体是针对类型 IIA MyHCs13进行探测的。利用我们的原始半自动算法, 利用几种图像变换从明图像中生成概率映射。从图 3a中所示的明图像导出的典型图像转换在图 3B中显示。利用斐济的变换和孔隙分析函数, 我们测量了平均 CSA (891.4 ±45.0 µm2), 最大值 (46.9 ±1.3 µm) 和最小值 (26.0 ±0.8 µm) 平均 Feret 直径。为了测试我们测量的精确度, 我们还手动评估了明图像中的这些参数。平均 CSA (867.0 ±52.0 µm2), 最大值 (45.7 ±3.5 µm) 和最小值 (25.6 ±1.9 µm) 平均 Feret 直径获得使用更繁琐的手工采集方法是没有明显不同的半自动方法 (所有的p和 #62;0.05, 不成对的t-测试;表 3)。
<img alt="Table 3" src alt="Table 3"></img alt="Table 3" src>= "/文件/ftp_upload/56024/56024table3. jpg"/> 
表 3: 由人工和半自动分析方法确定的 I 型、IIA、IIB 和 IIX MyHCs 胫骨前肌 immunostained 的纤维形态和组成.平均纤维 CSA, 平均 Feret 直径和 MyHC 类型的小鼠胫骨前量化通过传统的手工评估与提出的算法。请注意, 组合分数超过1的值, 因为存在的纤维表示一个以上的 MyHC 类型。
图 3C显示同一字段 immunostained 的荧光图像, 其抗体指向 IIA 型 MyHCs。用数字 (图 3D) 选择字段中的阳性染色纤维, 并通过除以明变换域中的纤维总数 (表 3) 来确定 IIA 型 MyHC 表达纤维的分数。).然后评估了 a 型 IIA 纤维的平均 CSA、最大和最小 Feret 直径 (表 3)。I 型, 类型 IIX 和类型 IIB 纤维的比例, 以及它们的尺寸, 是通过在 immunostained 与 BA-F8, 6H1 和 BF-F3 抗体, 分别6,12(表 4) 中重复此协议而确定的。;图 4A-d)。
<img alt="Table 4" src="/files/ftp_upload/56024/56024table4.jpg"> 
表 4: 小鼠比目鱼肌和胫骨前 MyHC 的含量和形态学.比目鱼肌和胫骨前部的纤维型成分。显示了比目鱼肌和胫骨前 Feret 的平均 CSA、最小和最大的 i 型、a 型 IIA、型 IIX 和型 IIB 的定量。数据以平均±SEM。
我们还评估了这些参数在一个领域获得的比目鱼肌 (表 4;图 4A-d)。分别对胫骨前和比目鱼肌的3052和2876个别纤维进行计数。同时, 这些数据证明了我们的方法在纤维型和形态分析中的可行性。
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56024/56024fig4.jpg"> 
图 4: 形态分析数据汇总.纤维型成分的代表性部分的胫骨前和比目鱼肌 (A)。在上班族胫骨和比目鱼中, i 型、a 型 IIA、IIX 型和型 IIB anteriort 的平均 CSA、minimimal 和最大 Feret 直径的量化, 如B-D所示。数据是平均±SEM. 胫骨前和比目鱼肌之间有显著差异 (* 表示p和 #60; 0.05: ** 表示p和 #60; 0.01; ** 表示p和 #60; 0.005; t 测试)。分别对胫骨前和比目鱼肌的3052和2876个别纤维进行计数。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56024/56024fig4large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Supplemental File" src="/files/ftp_upload/56024/56024supfile.jpg"> 
补充文件: 光纤量化宏.<a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56024/Tyagi_etal_fiber_quantification_macro" target="_blank">请单击此处下载此文件.</a>

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

Nanyang Technological University

Research

JoVE Journal

Medicine

10.3K 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, ...

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

The percutaneous biopsy technique enables researchers and clinicians to collect skeletal muscle tissue samples. The technique is safe and highly effective. This video describes the percutaneous biopsy technique using a modified Bergstr&#246;m needle to obtain skeletal muscle tissue samples from the vastus lateralis of human subjects. The Bergstr&#246;m needle consists of an outer cannula with a small opening (&#8216;window&#8217;) at the side of the tip and an inner trocar with a cutting blade at the distal end. Under local anesthesia and aseptic conditions, the needle is advanced into the skeletal muscle through an incision in the skin, subcutaneous tissue, and fascia. Next, suction is applied to the inner trocar, the outer trocar is pulled back, skeletal muscle tissue is drawn into the window of the outer cannula by the suction, and the inner trocar is rapidly closed, thus cutting or clipping the skeletal muscle tissue sample. The needle is rotated 90&#176;&#160;and another cut is made. This process may be repeated three more times. This multiple cutting technique typically produces a sample of 100-200 mg or more in healthy subjects and can be done immediately before, during, and after a bout of exercise or other intervention. Following post-biopsy dressing of the incision site, subjects typically resume their activities of daily living right away and can fully participate in vigorous physical activity within 48-72 hr. Subjects should avoid heavy resistance exercise for 48 hr to reduce the risk of herniation of the muscle through the incision in the fascia.

The purpose of this video is to present the modified Bergstr&#246;m skeletal muscle biopsy technique on human subjects.

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

Quantitative magnetic resonance imaging (qMRI) describes the development and use of MRI to quantify physical, chemical, and/or biological properties of living systems. Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using qMRI methods. The MRI acquisition protocol begins with localizer images (used to locate the position of the body and tissue of interest within the MRI system), quality control measurements of relevant magnetic field distributions, and structural imaging for general anatomical characterization. The qMRI portion of the protocol includes measurements of the longitudinal and transverse relaxation time constants (T1 and T2, respectively). Also acquired are diffusion-tensor MRI data, in which water diffusivity is measured and used to infer pathological processes such as edema. Quantitative magnetization transfer imaging is used to characterize the relative tissue content of macromolecular and free water protons. Lastly, fat-water MRI methods are used to characterize fibro-adipose tissue replacement of muscle. In addition to describing the data acquisition and analysis procedures, this paper also discusses the potential problems associated with these methods, the analysis and interpretation of the data, MRI safety, and strategies for artifact reduction and protocol optimization.

Neuromuscular diseases often exhibit a temporally varying, spatially heterogeneous, and multi-faceted pathology. The goal of this protocol is to characterize this pathology using non-invasive magnetic resonance imaging methods.

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

Percutaneous muscle biopsy using the Weil-Blakesley conchotome is well established in both clinical and research practice. It is a safe, effective and well tolerated technique. The Weil-Blakesley conchotome has a sharp biting tip with a 4 - 6 mm wide hollow. It is inserted through a 5 - 10 mm skin incision and can be maneuvered for controlled tissue penetration. The tip is opened and closed within the tissue and then rotated through 90 -180&#176; to cut the muscle. The amount of muscle obtained following repeated sampling can vary from 20 mg to 290 mg which can be processed for both histology and molecular studies. The wound needs to be kept dry and vigorous physical activity kept to a minimum for approximately 72 hr although normal levels of activity can restart immediately following the procedure. This procedure is safe and effective when close attention is paid to the selection of subjects, full asepsis and post procedure care. &#160;Both right and left vastus lateralis are suitable for biopsy dependent on participant preference.

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

This video demonstrates the technique of percutaneous muscle biopsy of the human vastus lateralis 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

Contraction-induced muscle damage via voluntary eccentric (lengthening) contractions offers an excellent model for studying muscle adaptation and recovery in humans. Herein we discuss the design of an eccentric exercise protocol to induce damage in the quadriceps muscles, marked by changes in strength, soreness, and plasma creatine kinase levels. This method is simple, ethical, and widely applicable since it is performed in human participants and eliminates the interspecies translation of the results. Subjects perform 300 maximal eccentric contractions of the knee extensor muscles at a speed of 120&#176;/sec on an isokinetic dynamometer. The extent of the damage is measurable using relatively non-invasive isokinetic and isometric measures of strength loss, soreness, and plasma creatine kinase levels over several days following the exercise. Therefore, its application can be directed to specific populations in an attempt to identify mechanisms for muscle adaptation and regeneration.

This article describes a safe and reliable method to induce and quantify exertional skeletal muscle damage in human subjects.

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

An increasing number of genetically modified mouse models has become available in recent years. Moreover, the number of pharmacological studies performed in mice is high. Phenotypic characterization of these mouse models also requires the examination of cardiac function and morphology. Echocardiography and magnetic resonance imaging (MRI) are commonly used approaches to characterize cardiac function and morphology in mice. Echocardiographic and MRI equipment specialized for use in small rodents is&#160;expensive and requires a dedicated space. This protocol describes cardiac measurements in mice using a clinical echocardiographic system with a 15 MHz human vascular probe. Measurements are performed on anesthetized adult mice. At least three image sequences are recorded and analyzed for each animal in M-mode in the parasternal short-axis view. Afterwards, cardiac histological examination is performed, and cardiomyocyte diameters are determined on hematoxylin-eosin- or wheat germ agglutinin (WGA)-stained paraffin sections. Vessel density is determined morphometrically after Pecam-1 immunostaining. The protocol has been applied successfully to pharmacological studies and different genetic animal models under baseline conditions, as well as after experimental myocardial infarction by the permanent ligation of the left anterior descending coronary artery (LAD). In our experience, echocardiographic investigation is limited to anesthetized animals and is feasible in adult mice weighing at least 25 g.

Echocardiographic examination is frequently used in mice. Expensive high-resolution ultrasound devices have been developed for this purpose. This protocol describes an affordable echocardiographic procedure combined with histological morphometric analyses to determine cardiac morphology.

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

Alterations to body composition (fat or lean mass), metabolic parameters such as whole-body oxygen consumption, energy expenditure, and substrate utilization, and behaviors such as food intake and physical activity can provide important information regarding the underlying mechanisms of disease. Given the importance of body composition and metabolism to the development of obesity and its subsequent sequelae, it is necessary to make accurate measures of these parameters in the pre-clinical research setting. Advances in technology over the past few decades have made it possible to derive these measures in rodent models in a non-invasive and longitudinal fashion. Consequently, these metabolic measures have proven useful when assessing the response of genetic manipulations (for example knockout or transgenic mice, viral knock-down or overexpression of genes), experimental drug/compound screening and dietary, behavioral or physical activity interventions. Herein, we describe the protocols used to measure body composition and metabolic parameters using an animal monitoring system in chow-fed and high fat diet-fed mice.

This protocol describes the use of a body composition analyzer and metabolic animal monitoring system to characterize body composition and metabolic parameters in mice. An obesity model induced by high-fat feeding is used as an example for the application of these techniques.

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

Atherosclerosis remains the leading cause of death worldwide and, despite countless preclinical studies describing promising therapeutic targets, novel interventions have remained elusive. This is likely due, in part, to a reliance on preclinical prevention models investigating the effects of genetic manipulations or pharmacological treatments on atherosclerosis development rather than the established disease. Also, results of these studies are often confounding because of the use of superficial lesion analyses and a lack of characterization of lesion cell populations. To help overcome these translational hurdles, we propose an increased reliance on intervention models that employ investigation of changes in cellular composition at a single cell level by immunofluorescent staining and confocal microscopy. To this end, we describe a protocol for testing a putative therapeutic agent in a murine intervention model including a systematic approach for animal dissection, embedding, sectioning, staining, and quantification of brachiocephalic artery lesions. In addition, due to the phenotypic diversity of cells within late-stage atherosclerotic lesions, we describe the importance of using cell-specific, inducible lineage tracing mouse systems and how this can be leveraged for unbiased characterization of atherosclerotic lesion cell populations. Together, these strategies may assist vascular biologists to more accurately model therapeutic interventions and analyze atherosclerotic disease and will hopefully translate into a higher rate of success in clinical trials.

We propose a standardized protocol to characterize the cellular composition of late-stage murine atherosclerotic lesions including systematic methods of animal dissection, tissue embedding, sectioning, staining, and analysis of brachiocephalic arteries from atheroprone 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 ...