我们感激 Clemire Cimper 的出色技术支持。在实验室工作的资金由巴斯德研究所, 中心全国研究科学, 和国家研究 (Laboratoire 卓越复兴, Investissement d 艾文莉;ANR-10-LABX-73), 研究 (ANR-16-CE13-0017-01) 和基金会弧 (PJA 20161205028)。cc 和 ac 是由复兴财团的博士和博士后奖学金资助的。

动物按照欧洲共同体的准则和巴斯德研究所 (CETEA) 批准的议定书的伦理委员会处理. 
 1. 库存解决方案的准备工作 <ol> <li> 准备用于肌肉标本固定的材料。将0.5 克的粉胶与20毫升的水溶于 RT, 用于肌肉固定的冷冻埋入培养基. </li>
	<li> 准备 SA 和 #946 的解决方案;-Gal 染色. 
	<ol> <li> 通过在蒸馏水中溶解各自的粉末, 准备 k 3 Fe (cn) 6 (100 mm)、k 4 Fe (CN) 6 (100 mm)、氯化镁 2 (1M) 的库存解决方案. </li>
		<li> 准备 0.2% C 的库存解决方案 20 H 6 Br 4 Na 2 O 5 (曙红) 稀释它在水中。
		<li> 准备 c 14 H 15 BrClNO 6 (x gal) (50 毫克/毫升) 的库存解决方案, 方法是在 c 3 H 7 NO (胺, DMF) 中溶解 X gal 粉末. </li>
		<li> 存储 K 3 fe (cn) 6 和 k 4 fe (cn) 6 解决方案位于4和 #176; C 和氯化镁 2 在 RT 上. 
		注意: X gal 可存储在分-20 和 #176; C, 最多6月。红红溶液可在 RT 中保存, 必要时可在过滤后再使用。k 3 Fe (cn) 6 和 k 4 Fe (cn) 6 解决方案是 protectedfromthe 光。X gal 解决方案在水中不稳定, 必须保护不受光照. </li>
	</ol> </li> <li> Nanog 染色解决方案的准备: 性解决方案包含 0.1% Na 3 c 6 h 5 o 7 (柠檬酸三钠), 0.1% c 14 h 22 o (c 2 h 4 O) n (n = 9-10) (海卫 X-100) 在蒸馏水中, 应储存在4和 #176; c. 制备含有5% 胎牛血清 (FBS) 的含磷酸盐缓冲盐水 (PBS) 的阻断溶液, 应在 RT 中保存. </li>
</ol> 2。SA 和 #946;-Gal 染色在冰冻的 ta 肌肉部分 <ol> <li> 为 TA 肌肉准备固定材料, 将少量的粉胶放在软木片上. </li>
	<li> 受伤的小鼠 (2 月大, C57/B6) 在10天前与心脏 (CDX) 一起受伤, 如前面所描述的 13 。使用受害 (PBS 注入) TA 从同一个鼠标的负控制。如果 在体内 需要重新编程, 则在 Dox (1 毫克/毫升) 在饮用水中的同一天 (保护免受光照), 在酰损伤后. 
	注: Dox 解决方案需要每3天更换一次, 总处理时间为7天。
	<ol> <li> 将两个 TA 肌肉 (受伤和控制) 从小鼠中分离出来, 如前所述 ( 图 1A ) 13 。为了确保横断面, 将 TA 肌肉的远端肌腱插入粉胶中, 并 #190; 部分肌肉外 ( 图 1B ), 并直接冻结在液氮冷却戊的 #60; 1最小 
		注意: 确保 TA 肌肉处于垂直位置, 并在软木塞的中心。样品可存储在-80 和 #176; C 或直接 cryosectioned 在10和 #181; m 部分. </li>
	</ol> </li> <li> 处理如 图 1B 中所述的 TA 肌肉。
	<ol> <li> 将 1 st -10 th 节按正确的顺序分发到每张幻灯片左上角的十张不同的幻灯片上。将 11 th 节放在第一张幻灯片的 1 st 部分的旁边;除第二张幻灯片上的 2 nd 部分外, 12 th 节将按照相同的顺序放置。重复此过程直到10节/幻灯片获得十张幻灯片 (总共100节), 以确保第一节和最后一节之间的最小 1 mm 距离. </li>
	</ol> </li> <li> 在 PBS 中固定4分钟, 其中包含1% 甲醛和0.2% 戊二醛。用 PBS、2x 和 #160 冲洗; 10 分钟接下来, 在 PBS (pH = 5.5) 中孵育30分钟的部分, 执行 RT 中的所有步骤. 
	注意: 固定必须是温和的, 以保持酶活性。在引擎盖下面执行此步骤。PBS 的 pH 值是至关重要的, 必须保护 X gal 解决方案免受光照. </li>
	<li> 在 x gal 解决方案中孵育部分, 其中包含: 4 mm K3Fe (cn) 6 、4 mm K4Fe (cn) 6 、2 mm MgCl2 和400和 #181; PBS 中的 g/mL X gal, pH = 5.5。在黑暗中孵育37和 #176; C 至少24小时. 用 PBS 冲洗幻灯片, 3x 10 分钟, 在 RT 上. 
	注意: 孵化需要至少24小时, 并可持续48小时, 以最大化 SA 和 #946;-Gal 信号。该解决方案需要在24小时的孵化后改变。应保护幻灯片不受光照。如果只需要 SA 和 #946;-Gal 染色, 请继续执行步骤2.5。如果需要 co-staining 与 Nanog, 请跳到步骤 3.1. </li>
	<li> 在 pbs 中修复1% 甲醛中的幻灯片, 用 pbs 进行30分钟的洗涤, 3x 10 min. Counterstain 与0.2% 曙红。将这些幻灯片浸泡在红红溶液中1分钟, 并简单地用蒸馏水冲洗。最后, 用水性 non-fluorescing 安装介质安装幻灯片 (请参阅 材料表 ). 
	注意: 必须执行 post-fixation。在引擎盖下面执行此步骤。所有步骤都在 RT 上执行. </li>
</ol> 3。抗 Nanog 抗体的免疫组织化学研究 <ol> <li> 将带有 pbs 的4% 甲醛的幻灯片修复为 10 min. 2x 10 分钟添加200和 #181; 性溶液直接放在幻灯片上, 孵育5分钟。 <ol> <li> 用 pbs 清洗幻灯片, 2x 5 分钟, 最后一次洗涤, 使用200和 #181; L PBS 在幻灯片上直接包含 0.25% BSA。在 RT 上执行所有这些步骤. 
		注意: 在引擎盖下执行固定步骤. </li>
	</ol> </li> <li> 将具有主要抗 Nanog 抗体的幻灯片 (最终浓度: 1.25 微克/毫升) 过夜于4和 #176; PBS 中含有 5% FBS。用 pbs 清洗幻灯片, 2x 10 分钟, 最后一次洗涤, 使用200和 #181; L PBS 含有 0.25% BSA, 用于5分钟. 在 RT 上执行所有洗涤步骤. 
	注: 用湿纸巾在盒子里孵育幻灯片, 以防蒸发. </li>
	<li> 孵育100和 #181 的幻灯片; 从准备使用的工具包中的二次抗体的 L, (请参见 材料表 ) 为45分钟, 用 PBS 冲洗幻灯片, 3x 5 分钟, 去除二次抗体。在 RT 中执行所有步骤, 保护不受光照. </li>
	<li> 可视化 <ol> <li> 首先, 稀释33和 #39;-diaminobenzidine (c 12 h 14 N 4 c 12 h 18 Cl 4 N 4 4 HCl) 在基板缓冲区中由准备使用的工具包 (20 和 #181; L 为1毫升的基板缓冲液) 提供。在每张幻灯片上直接添加100和 #181, 并在 RT 中孵育最多10分钟. 
		注: 稀释后的民建联解决方案应该是新鲜的, 可以在4和 #176 的一周内储存。孵化时间可以调整到最小化背景信号, 但必须保持相同的所有幻灯片. </li>
		<li> 通过用水冲洗消除民建联的解决方案。Counterstain 快速红色解决方案 (准备使用, 请参阅 材料表 ) 的幻灯片, 20 分钟. 简要地再用水洗涤幻灯片. </li>
		<li> 脱水他们与95% 乙醇为 5 m其次是100% 乙醇, 2x 5 分钟。最后, 用快速硬化安装介质装载幻灯片 (请参阅 材料表 ). </li>
		<li> 在20X 的明亮场中观察显微镜下的幻灯片以避免背景. 
		注: 快速红色溶液可在 RT 中保存并在过滤后重复用. </li>
	</ol> </li> </ol> 4。分析和量化 <ol> <li> SA 和 #946;-Gal 量化 <ol> <li> 扫描幻灯片, 并根据获得的图像选择每个幻灯片上的最佳分区。使用至少2节/TA 的量化。根据组织完整性、染色质量和 counterstaining, 判断切片和 #39 质量。对于量化, 选择两个最高质量的部分, 最大可能的距离在样品之间, 这使得 SA 和 #946 更好的代表性;-Gal 量化整个 TA 肌肉. </li>
		<li> 量化 SA 和 #946;-Gal 阳性单元格 ( 图 2 )。
		<ol> <li> 通过手动选择最小和最大的正向 SA 和 #946;-Gal 单元格来确定像素大小的秩。
			<ol> <li> 使用 ImageJ 软件打开扫描的幻灯片的数字图像. </li>
				<li> 在界面中, 单击并 #39; 分析、#39、#62、#39、工具、#39、#62、#39、#39、#62、#39、#39、#62、#39、#39、#62 等; 选择区域和 #39;使用选择工具, 并围绕最小和最大的积极 SA 和 #946;-Gal 单元格, 并通过单击和 #39 将其添加到 ROI 管理器; 添加和 #39;。使用 and #39 测量大小; 测量和 #39; 按钮并保存这些值以备以后使用. </li>
			</ol> </li> <li> 调整阈值参数以确保选中所有可见的正单元格。
			<ol> <li> 通过单击和 #39; 图像和 #39; #62; #39; 类型和 #39; #62; #39; 8 位和 #39; ( 图 2A ), 将图像转换为灰度级。接下来, 去 #39; 图像和 #39; #62; #39; 调整 #39; #62; #39; 阈值和 #39;, 移动第二个游标, 直到所有正的 SA 和 #946;-Gal 单元格以红色覆盖 ( 图 2B ), 然后单击和 #39; 应用和 #39; </li>
				<li> 通过单击和 #39 分析粒子; 分析和 #39; #62; #39; 分析粒子和 #39; #62; #39; 大小 (pixel^2) 和 #39;, 并应用在步骤4.1.2.1 中定义的值, 输入和 #39; 循环和 #39;: #39; 0.00-1. 00 和 #39;, 单击和 #39; ok 和 #39;; 所有的摘要计数的粒子显示在 ROI 管理器中 ( 图 2D ). </li>
			</ol> </li> <li> 将所有选定的粒子转移到原始图像。手动调整选择以确保精确的量化。添加正单元格 (绿色箭头, 图 2E ) 和/或删除假阳性单元格 (红色箭头, 图 2E )。最后, 通过使用 and #39 对区域 ( 图 2D ) 进行分级, 从而测量其面积; 选择工具和 #39;。单击和 #39; ROI 管理器和 #39; #62; #39; 添加和 #39; #62; #39; 度量和 #39;. </li>
			<li> 重要的是, 按节的区域规范化正单元格的数目. </li>
		</ol> </li> </ol> </li> <li> Nanog 量化 <ol> <li> 在明亮的字段中, 在显微镜下以20X 倍的比例手动计数 Nanog 阳性细胞. 
		注: 快速红 counterstaining 可以很好地评价 TA 肌肉的形态。然而, 染色是非常轻的, 缺乏一个明确的部分轮廓。扫描仪通常无法识别剖面的边界, 无法正确对焦。在扫描之前, 可以使用标记来圈出剖面的边缘, 或者使用更灵敏的扫描仪来检测剖面。对于当前协议, 最有效的方法是手动计算 Nanog 的正单元格. </li>
	</ol>
	</li>
</ol>

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作者声明他们没有竞争的金融利益。

在这里, 我们提出了一种检测编程小鼠骨骼肌中的衰老和多能干细胞的方法。该方法可用于评价和量化衰老和诱导细胞可塑性在体内, 并检查衰老在组织修复和再生中的作用。
在目前的协议, 衰老相关的β-乳糖 (SA β加仑) 检测被用来探测体内衰老细胞的骨骼肌肉。这种检测检测到增加的溶酶体β-乳糖活性在 suboptimum ph 值 (6.0 或 5.5), 特别是与衰老细胞相关, 而这酵?...

细胞衰老是一种以稳定的细胞周期阻滞为特征的应激反应形式。在过去的十年中, 研究已经确定衰老与各种生物学和病理过程有关, 包括胚胎发育、纤维化和生物体老化1,2。细胞衰老首先被确定在人成纤维细胞在其复制寿命结束时由端粒缩短3触发。除了复制的压力, 还有许多其他刺激, 可以诱导衰老, 包括 DNA 损伤, 氧化应激, 致癌信号, 和基因组/表的改变, 其中任何可能最终激活 p53/p21 和/或 pRB 途径建立并加强永久性增长逮捕1。衰老细胞的一个重要特征是它们保持新陈代谢活性, 并表现出衰老相关的分泌表型 (SASP): 许多炎症细胞因子、生长因子和细胞外基质的分泌因素4。SASP 因子在调节和增强衰老作用方面起着重要作用, 因为它们对吸引免疫细胞和改变局部和全身组织环境1有很强的影响。有趣的是, 衰老最近被提出是重要的组织修复和再生5,6。此外, 包括我们在内的几个实验室的数据表明, 组织损伤导致的衰老可能增强细胞的可塑性, 通过 SASPs, 促进再生7-9。因此, 所有涌现的数据突出了研究衰老的重要性在体内。
在后诱导多能干细胞 (iPSC) 时代, 细胞可塑性是一个细胞获得一个新的身份和采取替代的命运时, 暴露在不同的刺激, 在文化和在体内10的能力。众所周知, 完全重新编程可以实现在体内11,12, 其中包含四 Yamanaka 因素的卡带的表达: Oct4, Sox2, Klf4, 和c-myc(OSKM) 可诱导体内促进多器官畸胎瘤的形成。因此, 一个编程的小鼠模型 (i4F) 可以被用来作为一个强大的系统, 以确定关键的调节器和路径, 是重要的细胞可塑性11。
一个合适的和敏感的在体内系统是必要的, 以了解如何细胞衰老调节细胞可塑性的背景下组织再生。在这里, 我们提出了一个健壮的系统和详细的协议, 以评估在组织再生的背景下衰老和细胞可塑性之间的联系。心脏 (酰) 诱导的肌肉损伤的胫骨前 (TA) 肌肉组, 一个行之有效的系统研究组织再生, 和 i4F 小鼠模型, 允许检测细胞衰老和在体内在肌肉再生过程中重新编程。
为了评估细胞可塑性和衰老之间的联系, i4F 小鼠受酰损伤, 用强力霉素 (0.2 毫克/毫升) 在7天内诱发急性肌肉损伤, 以诱导体内重编程。虽然酰引起的急性肌肉损伤和再生协议最近已发布13, 出于道德上的原因, 此过程将在当前协议中省略。TA 肌肉样本将收集在10天后受伤的13, 当衰老细胞的峰值已经被观察到之前,14。在这里, 这个详细的协议描述了所有的步骤来评估衰老的水平 (通过 SA β-加仑) 和重新编程 (通过 IHC 染色 Nanog)。
衰老相关的β-乳糖 (SA-β-加仑) 检测是最常用的检测方法, 可以在培养和体内15中发现衰老细胞。与其他检测方法相比, SA β-加仑法可以在其本机环境中使用完整的组织结构来鉴定衰老细胞, 这对体内研究尤为重要。此外, 它是可能的夫妇的 SA β加仑测定与其他标记使用 IHC。然而, SA β-加仑检测确实需要新鲜或冰冻样本, 这仍然是一个主要的限制。当新鲜或冰冻的组织是常规可用的, 如冰冻的 TA 肌肉样本, SA β-加仑显然是最合适的检测衰老细胞的方法。Nanog 是用于检测 reprogramed 细胞的标记, 有两个原因: 1) 是多的重要标志;2) 更重要的是, 它的表达不是由强力霉素 (dox) 驱动的, 因此它检测诱导的多而不是 Yamanaka 盒的强迫表达。
值得注意的是, 本研究中提出的染色协议可以单独进行, 以简化量化程序, 但也可以在 co-staining 过程中进行, 以在同一剖面上显示衰老和多能干细胞。

<table>
	<tbody>
		<tr>
			<td>K3Fe(CN)6</td>
			<td>Sigma</td>
			<td>13746-66-2</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>K4Fe(CN)6</td>
			<td>Sigma</td>
			<td>14459-95-1</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>MgCl2</td>
			<td>Sigma</td>
			<td>7786-30-3</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>X-Gal</td>
			<td>Sigma</td>
			<td>B4252</td>
			<td>For SA-&#946; Gal staining solution</td>
		</tr>
		<tr>
			<td>Doxycycline</td>
			<td>Sigma</td>
			<td>D3447</td>
			<td>For inducing in vivo reprogramming</td>
		</tr>
		<tr>
			<td>Cardiotoxin</td>
			<td>Lotaxan Valence, France</td>
			<td>L8102</td>
			<td>For muscle injury</td>
		</tr>
		<tr>
			<td>Glutaraldehyde</td>
			<td>Sigma</td>
			<td>111-30-8</td>
			<td>For Fixation solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Electron microscopy science</td>
			<td>50-980-487</td>
			<td>For Fixation solution</td>
		</tr>
		<tr>
			<td>NaCitrate : Sodium Citrate monobasic bioxtra, anhydre</td>
			<td>Sigma</td>
			<td>18996-35-5</td>
			<td>For permeabilization solution</td>
		</tr>
		<tr>
			<td>Triton</td>
			<td>Sigma</td>
			<td>93443</td>
			<td>For permeabilization solution</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma</td>
			<td>A3608</td>
			<td>Washing solution</td>
		</tr>
		<tr>
			<td>Antibody anti- Nanog</td>
			<td>Cell signalling</td>
			<td>8822S</td>
			<td>Rabbit monoclonal antibody</td>
		</tr>
		<tr>
			<td>EnVision+ Kits (HRP. Rabbit. DAB+)</td>
			<td>Dako</td>
			<td>K4010</td>
			<td>For Nanog revelation</td>
		</tr>
		<tr>
			<td>Eosin 1%</td>
			<td>Leica</td>
			<td>380159EOF</td>
			<td>Counterstainning</td>
		</tr>
		<tr>
			<td>Fast red</td>
			<td>Vector Laboratories</td>
			<td>H-3403</td>
			<td>Counterstainning</td>
		</tr>
		<tr>
			<td>Thermo Scientific Shandon Immu-Mount</td>
			<td>Fisher scientific</td>
			<td>9990402</td>
			<td>Mounting solution</td>
		</tr>
		<tr>
			<td>Quick-hardening mounting medium for microscopy : Eukitt&#174;</td>
			<td>Sigma</td>
			<td>25608-33-7</td>
			<td>Mounting solution</td>
		</tr>
		<tr>
			<td>Microscope Phase Contrast Brightfield CKX41: 10X-20X-40X objectives</td>
			<td>Olympus</td>
			<td>CKX41</td>
			<td>Microscope for Nanog quantification</td>
		</tr>
		<tr>
			<td>Mouse: i4F-A</td>
			<td>Abad et al., 2013</td>
			<td>N/A</td>
			<td>Reprogrammable mouse model</td>
		</tr>
		<tr>
			<td>Skeletal muscle, Tibialis Anterior</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Slide Scanner</td>
			<td>Zeiss</td>
			<td>Axio Scan Z1</td>
			<td>slides scanning</td>
		</tr>
	</tbody>
</table>

evaluation of injury-induced senescence and in vivo reprogramming in the skeletal muscle

细胞衰老是一种应激反应, 其特点是稳定的细胞生长阻滞, 这是重要的许多生理和病理过程, 如癌症和衰老。最近, 衰老也牵涉到组织修复和再生。因此, 识别衰老细胞在体内变得越来越重要。衰老相关的β-乳糖 (SA β-加仑) 检测是最广泛使用的检测方法, 在培养和体内的衰老细胞。这种方法是基于在衰老细胞中增加溶酶体的含量, 这使得在 suboptimum pH 值 (6 或 5.5) 上, 组织化学检测溶体乳糖活性。与其他的检测方法, 如流式细胞仪, 这使得在其居住环境中的衰老细胞的鉴定, 这提供了宝贵的信息, 如与组织结构的位置, 形态学, 和通过免疫组织化学 (IHC) 与其他标记物耦合的可能性。对新鲜的或冰冻的样品的要求是对 SA β-加仑测定的主要限制。
在这里, 我们提出了一个详细的协议, 以了解如何细胞衰老促进细胞可塑性和组织再生在体内。我们使用 SA β-加仑来检测骨骼肌损伤后的衰老细胞, 这是一个行之有效的组织再生研究系统。此外, 我们使用 IHC 检测 Nanog, 一个多能干细胞的标志, 在转基因小鼠模型。该协议使我们能够检查和量化细胞衰老的背景下诱导细胞可塑性和体内重新编程。

肌损伤诱导细胞衰老的检测
最近已经证明, 肌肉损伤诱发短暂的细胞衰老14。在10天后 (DPI), 大部分受损的纤维正在进行再生与中央定位核, 一个标志, 再生纤维, 并重建肌肉的结构。在某些地区, 浸润性炎症细胞明显减少, 但仍可见。10 DPI 是一个很好的时间点来检测衰老细胞的 SA β-Gal, 因为有较少的坏死和炎症细?...

Watch this Scientific Journal Video about Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle at JoVE.com

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

骨骼肌损伤诱导衰老和体内重编程的评价

在这里, 我们提出了一个详细的协议, 以检测两个衰老和多能干细胞的骨骼肌肉损伤时, 诱导在体内重新编程。该方法适用于评价细胞衰老在组织再生过程中的作用和重新编程体内。

Cellular senescence is a stress response that is characterized by a stable cellular growth arrest, which is important for many physiological and ...

evaluation-injury-induced-senescence-vivo-reprogramming-skeletal

Research

JoVE Journal

Developmental Biology

Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

9.5K 次观看.  Institut Pasteur. 在这里, 我们提出了一个详细的协议, 以检测两个衰老和多能干细胞的骨骼肌肉损伤时, 诱导在体内重新编程。该方法适用于评价细胞衰老在组织再生过程中的作用和重新编程体内。

视频: Evaluation of Injury-induced Senescence and In Vivo Reprogramming in the Skeletal Muscle

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

斑马鱼幼虫骨骼肌诚信与伊文思蓝分析

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

科研

发育生物学

Using Isolated Mitochondria from Minimal Quantities of Mouse Skeletal Muscle for High throughput Microplate Respiratory Measurements

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of mitochondrial function in this tissue has become more prevalent. Although many technologies and assays exist that measure mitochondrial respiratory pathways in a variety of cells, tissue and species, there is currently a void in the literature in regards to the compilation of these assays using isolated mitochondria from mouse skeletal muscle for use in microplate based technologies. Importantly, the use of microplate based respirometric assays is growing among mitochondrial biologists as it allows for high throughput measurements using minimal quantities of isolated mitochondria. Therefore, a collection of microplate based respirometric assays were developed that are able to assess mechanistic changes/adaptations in oxygen consumption in a commonly used animal model. The methods presented herein provide step-by-step instructions to perform these assays with an optimal amount of mitochondrial protein and reagents, and high precision as evidenced by the minimal variance across the dynamic range of each assay.

The methods presented provide step-by-step instructions for the performance of a collection of microplate based respirometric assays using isolated mitochondria from minimal quantities of mouse skeletal muscle. These assays are able to measure mechanistic changes/adaptations in mitochondrial oxygen consumption in a commonly used animal model.

使用分离线粒体从小鼠骨骼肌进行高通量微孔板呼吸测量最小数量

Skeletal muscle mitochondria play a specific role in many disease pathologies. As such, the measurement of oxygen consumption as an indicator of ...

Induction of Acute Skeletal Muscle Regeneration by Cardiotoxin Injection

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced skeletal muscle regeneration is a widely used, powerful model system to study the events involved in muscle regeneration as well as the mechanisms and different players. Indeed, a detailed knowledge of this process is essential for a better understanding of the pathological conditions that lead to skeletal muscle degeneration, and it aids in identifying new targeted therapeutic strategies. The present work describes a detailed and reproducible protocol to induce acute skeletal muscle regeneration in mice through a single intramuscular injection of cardiotoxin (CTX). CTX belongs to the family of snake venom toxins and causes myolysis of myofibers, which eventually triggers the regeneration events. The dynamics of skeletal muscle regeneration is evaluated by histological analysis of muscle sections. The protocol also illustrates the experimental procedures for dissecting, freezing, and cutting the Tibialis Anterior muscle, as well as the routine Hematoxylin &#38; Eosin staining that is widely used for subsequent morphological and morphometric analysis.

This manuscript describes a detailed protocol to induce acute skeletal muscle regeneration in adult mice and subsequent manipulations of the muscles, such as dissection, freezing, cutting, routine staining, and myofiber cross-sectional area analysis.

通过注射心脏毒素急性骨骼肌再生的诱导

Skeletal muscle regeneration is a physiological process that occurs in adult skeletal muscles in response to injury or disease. Acute injury-induced ...

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the proteins involved in these pathways are evolutionarily conserved between mammals and other eukaryotes, such as the fruit fly Drosophila melanogaster, suggesting that similar organizing principles exist during the development of these organisms. Importantly, Drosophila has been used extensively to identify cellular and molecular mechanisms regulating processes that are required in mammals including neurogenesis, differentiation, axonal guidance, and synaptogenesis. Flies have also been used successfully to model a variety of human neurodevelopmental diseases. Here we describe a protocol for the step-by-step microdissection, fixation, and immunofluorescent localization of proteins within the adult Drosophila brain. This protocol focuses on two example neuronal populations, mushroom body neurons and retinal photoreceptors, and includes optional steps to trace individual mushroom body neurons using Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. Example data from both wild-type and mutant brains are shown along with a brief description of a scoring criteria for axonal guidance defects. While this protocol highlights two well-established antibodies for investigating the morphology of mushroom body and photoreceptor neurons, other Drosophila brain regions and the localization of proteins within other brain regions can also be investigated using this protocol.

Dissection and Immunofluorescent Staining of Mushroom Body and Photoreceptor Neurons in Adult Drosophila melanogaster Brains

This protocol describes the dissection and immunostaining of adult Drosophila melanogaster brain tissues. Specifically, this protocol highlights the use of Drosophila mushroom body and photoreceptor neurons as example neuronal subsets that can be accurately used to uncover general principles underlying many aspects of neuronal development.

成人果蝇脑内蘑菇体和感光神经元的解剖和荧光染色

Nervous system development involves a sequential series of events that are coordinated by several signaling pathways and regulatory networks. Many of the ...

Application of Chronic Stimulation to Study Contractile Activity-induced Rat Skeletal Muscle Phenotypic Adaptations

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To investigate the underlying mechanisms that bring about various muscle adaptations, a number of exercise protocols such as treadmill, wheel running, and swimming exercise have been used in the animal studies. However, these exercise models require a long period of time to achieve muscle adaptations, which may be also regulated by humoral or neurological factors, thus limiting their applications in studying the muscle-specific contraction-induced adaptations. Indirect low frequency stimulation (10 Hz) to induce chronic contractile activity (CCA) has been used as an alternative model for exercise training, as it can successfully lead to muscle mitochondrial adaptations within 7 days, independent of systemic factors. This paper details the surgical techniques required to apply the treatment of CCA to the skeletal muscle of rats, for widespread application in future studies.

This protocol describes the use of the chronic contractile activity model of exercise to observe stimulation-induced skeletal muscle adaptations in the rat hindlimb.

慢性刺激在收缩活动性大鼠骨骼肌表型适应性研究中的应用

Skeletal muscle is a highly adaptable tissue, as its biochemical and physiological properties are greatly altered in response to chronic exercise. To ...

Identification of Skeletal Muscle Satellite Cells by Immunofluorescence with Pax7 and Laminin Antibodies

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, antibodies for specific cell markers are applied on tissue sections. In adult skeletal muscle, satellite cells (SCs) are stem cells that contribute to muscle repair and regeneration. Therefore, it is important to visualize and trace the satellite cell population under different physiological conditions. In resting skeletal muscle, SCs reside between the basal lamina and myofiber plasma membrane. A commonly used marker for identifying SCs on the myofibers or in cell culture is the paired box protein Pax7. In this article, an optimized Pax7 immunofluorescence protocol on skeletal muscle sections is presented that minimizes non-specific staining and background. Another antibody that recognizes a protein (laminin) of the basal lamina was also added to help identify SCs. Similar protocols can also be used to perform double or triple labeling with Pax7 and antibodies for additional proteins of interest.

The precise identification of satellite cells is essential for studying their functions under various physiological and pathological conditions. This article presents a protocol to identify satellite cells on adult skeletal muscle sections by immunofluorescence-based staining.

Pax7 和层粘连蛋白抗体免疫荧光法鉴定骨骼肌卫星细胞

Immunofluorescence is an effective method that helps to identify different cell types on tissue sections. In order to study the desired cell population, ...

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from animals to humans. Therefore, the powerful Drosophila model system can be used to study concepts of muscle-tendon development that can also be applied to human muscle biology. Here, we describe in detail how morphogenesis of the adult muscle-tendon system can be easily imaged in living, developing Drosophila pupae. Hence, the method allows investigating proteins, cells and tissues in their physiological environment. In addition to a step-by-step protocol with helpful tips, we provide a comprehensive overview of fluorescently tagged marker proteins that are suitable for studying the muscle-tendon system. To highlight the versatile applications of the protocol, we show example movies ranging from visualization of long-term morphogenetic events &#8211; occurring on the time scale of hours and days &#8211; to visualization of short-term dynamic processes like muscle twitching occurring on time scale of seconds. Taken together, this protocol should enable the reader to design and perform live-imaging experiments for investigating muscle-tendon morphogenesis in the intact organism.

In Vivo Imaging of Muscle-tendon Morphogenesis in Drosophila Pupae

Here, we present an easy-to-use and versatile method to perform live imaging of developmental processes in general and muscle-tendon morphogenesis in particular in living Drosophila pupae.

在体内果蝇蛹肌肌腱形态的影像学研究

Muscles together with tendons and the skeleton enable animals including humans to move their body parts. Muscle morphogenesis is highly conserved from ...

Induction and Validation of Cellular Senescence in Primary Human Cells

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a hallmark of various pathophysiological conditions including tumor suppression, tissue remodeling and aging. The inducers of cellular senescence in vivo are still poorly characterized. However, a number of stimuli can be used to promote cellular senescence ex vivo. Among them, most common senescence-inducers are replicative exhaustion, ionizing and non-ionizing radiation, genotoxic drugs, oxidative stress, and demethylating and acetylating agents. Here, we will provide detailed instructions on how to use these stimuli to induce fibroblasts into senescence. This protocol can easily be adapted for different types of primary cells and cell lines, including cancer cells. We also describe different methods for the validation of senescence induction. In particular, we focus on measuring the activity of the lysosomal enzyme Senescence-Associated &#946;-galactosidase (SA-&#946;-gal), the rate of DNA synthesis using 5-ethynyl-2&#39;-deoxyuridine (EdU) incorporation assay, the levels of expression of the cell cycle inhibitors p16 and p21, and the expression and secretion of members of the Senescence-Associated Secretory Phenotype (SASP). Finally, we provide example results and discuss further applications of these protocols.

Here, we discuss a series of protocols for induction and validation of cellular senescence in cultured cells. We focus on different senescence-inducing stimuli and describe the quantification of common senescence-associated markers. We provide technical details using fibroblasts as a model, but the protocols can be adapted to various cellular models.

原发性细胞衰老的诱导与鉴定

Cellular senescence is a state of permanent cell cycle arrest activated in response to different damaging stimuli. Activation of cellular senescence is a ...

A Quantitative Measurement of Reactive Oxygen Species and Senescence-associated Secretory Phenotype in Normal Human Fibroblasts During Oncogene-induced Senescence

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. Recent studies have extended the role of cellular senescence to various physiological processes, including development, wound healing, immune surveillance, and age-related tissue dysfunction. Although cell cycle arrest is a critical hallmark of cellular senescence, an increased intracellular reactive oxygen species (ROS) production has also been demonstrated to play an important role in the induction of cellular senescence. In addition, recent studies revealed that senescent cells exhibit potent paracrine activities on neighboring cells and tissues through a senescence-associated secretory phenotype (SASP). The sharp increase in interest regarding therapeutic strategies against cellular senescence emphasizes the need for a precise understanding of senescence mechanisms, including intracellular ROS and the SASP. Here, we describe protocols for quantitatively assessing intracellular ROS levels during H-Ras-induced cellular senescence using ROS-sensitive fluorescent dye and flow cytometry. In addition, we introduce sensitive techniques for the analysis of the induction of mRNA expression and secretion of SASP factors. These protocols can be applied to various cellular senescence models.

Intracellular ROS has been shown to play an important role in the induction of cellular senescence. Here, we describe a sensitive assay for quantifying ROS levels during cellular senescence. We also provide protocols for assessing the senescence-associated secretory phenotype, which reportedly contributes to various age-related dysfunctions.

基因型衰老过程中正常人成纤维细胞活性氧和衰老相关分泌表型的定量测定

Cellular senescence has been considered a state of irreversible growth arrest upon exhaustion of proliferative capacity or exposure to various stresses. ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

用扫描电镜和全贴免疫荧光技术可视化小鼠胚胎中的节点和外型板

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...