资助离子和 RGB: 瑞士国家科学基金会 Ambizione-分数补助金 PZ00P3 142404/1 和 PZ00P3 163979。

所有实验都是按照帕特的声明进行的, 用于眼科和视力研究中的动物使用, 并得到地方当局的批准. 
 1. 在硅片上制备超薄切片 <ol> <li> 样品固定 <ol> <li> 准备含有0.1% 戊二醛的定影液, 4% 甲醛在0.1 米甲缓冲中. 
		注意: 小心! 在使用戊二醛、甲醛和甲缓冲液时要小心, 佩戴适当的个人防护设备, 并在通风罩中工作. </li>
		<li> 安乐5天后受精 (5 dpf) 斑马鱼幼虫与 tricaine (乙基 3-氨基甲基磺酸, 0.4% 瓦特/v 在 PBS (磷酸盐缓冲盐水, pH 7)), 如前所述 15 . </li>
		<li> 通过在冰上浸泡 pre-chilled 固定液来修复幼虫。取下头部, 在4和 #176 的夜间孵育; C 带轻柔摇摆. </li>
		通过1% 琼脂糖层板上的冷冻固定液, 用细钳和显微解剖刀 (在双目下) 修剪眼部周围的组织, <li> 解剖眼睛。用新鲜的 pre-chilled 固定剂把眼睛转到管子上. </li>
		<li> 在 PBS 中洗涤两次 (磷酸盐缓冲盐水, pH 值 7.4), 每次在室温下洗涤5分钟 (RT). </li>
	</ol> </li> <li> 明胶渗透和安装 <ol> <li> 将15毫升本地食品品牌明胶12% 瓦特/#160;(p hosphate 缓冲, 0.1 米 pH 7.4) 在40和 #176; c. 除去 PBS, 并在含有眼睛的管子上加入明胶溶液。轻轻拍打试管, 确保明胶渗入样品, 孵育 10-30 分钟, 在40和 #176; C 在 thermoblock 与温和晃动或在水浴. </li>
		<li> 填充 12 mm x 5 mm x 3 mm 硅或聚乙烯平面嵌入模具, 用温明胶在40和 #176; C 水浴。使用吸管在每个模具上加两只眼睛, 用解剖针在双目下正确对准, 让明胶在室温下冷却1分钟, 硬化4和 #176; C 为 20 min. </li>
		<li> 重新修剪在双目下的明胶块, 使用刀片式服务器每块用一只眼睛. </li>
		<li> 在冰上将明胶嵌入的眼睛转移到2.3 米的蔗糖中。在晚上4和 #176 孵育; C. </li>
		<li> 交换到新的2.3 米蔗糖溶液和存储在4和 #176; c 或-20 和 #176; c; 在这一步之后, 样品准备切片, 或者可以存储在-20 和 #176; c 几个星期到几个月. </li>
		<li> 将明胶块重新修整到几乎眼睛的大小, 然后再转到冷冻针。在液氮中冷冻并转移到低温 ultramicrotome. </li>
	</ol> </li> <li> Cryosectioning <ol> <li> 在-120 和 #176 处剪切 110 nm 厚剖面; C 在冷冻 ultramicrotome 中使用金刚石刀. </li>
		<li> 选取含有2% 纤维素 (在水中) 和2.3 米蔗糖溶液 (1:1) 的有线回路的截面。将截面转换为 7 mm x 7 mm 硅片。在4和 #176 中存储部分, 直到进一步处理. </li>
	</ol> </li> </ol> 2。Immunolabelling <ol> <li> 在0和 #176 上用 PBS 清洗硅片; C 为20分钟, 四和 #160;d 保护, 将晶片倒置放在水滴上。在室温下用 PBS 2x 冲洗2分钟. </li>
	<li> 在 PBS 中孵育3倍于0.15% 甘氨酸, 每1分钟。用 PBS 清洗 3x 1 分钟。前孵育与 PBG (PBS 与0.5% 牛血清白蛋白 BSA 和0.2% 明胶类型 B) 为 5 min. </li>
	<li> 在室温下用 PBG (4 和 #181、g/毫升) 的 anti-Tom20 (参见 材料表 ) 进行孵育30分钟. </li>
	<li> 洗 6x 1 分钟/PBG。孵育与 PBG 5 分钟在 RT. 孵育与兔 Alexa 647 F (ab 和 #39;) 2 (参见 材料目录 ) 在 PBG (7.5 和 #181; g/毫升) 为 30 min. </li>
	<li> 洗 6x 1 分钟/PBG。在 PBS 中洗 3x 2 分钟。在 pbs 中孵育 DAPI (4 和 #181; g/毫升), 十年代. 在 pbs 中冲洗 2x 2 分钟. </li>
</ol> 3。超分辨率显微术 <ol> <li> 通过将其放置在甘油 (80%) 的1:1 溶液的液滴和含有氧清除系统的成像缓冲器 (含10% 葡萄糖的200毫米磷酸缓冲液, 0.5 毫克/毫升glucoseoxidase, 40 和 #181; g/毫升过氧化氢酶, 15 毫米β-mercaptoethylamine 盐酸盐 (HCL), pH 值 8.0). </li>
	<li> 将晶圆片 (面朝下) 转移到玻璃底部 (厚度170和 #177; 5 和 #181; m) 培养皿放在甘油 (80%) 和成像缓冲器的1:1 混合物的新滴上 (如步骤 3.1). </li>
	<li> 从四面八方移除, 用吸管, 大部分的液体在晶片下面。使用硅胶条纹将晶片固定在培养皿的底部. 
	注: 硅胶条纹由双组分硅胶 (3 mm x 12 mm) 制成. </li>
	<li> 使用高数值孔径的倒置显微镜上的图像剖面 ( 如 160X 和 #160;/NA 1.43; 查看材料 表) 油浸超分辨率专用目标。在成像之前, 让样品平衡到显微镜温度, 以最小化/减少横向和轴向漂移. </li>
	<li> 将感兴趣的区域居中并获取第一个 widefield 荧光参考图像。更改为超分辨率操作模式。将相机的曝光时间调整为15毫秒, 并将电子倍增 (EM) 增益设置为最大值 300. </li>
	<li> 在荧光模式下用 642 nm 连续波激光照射最大激光功率 (对应于 ~ 2.8 kW/cm 2 ) 的样品。只要单分子闪烁在每个帧中都是很好的分离, 使单个信号重叠的概率较低, 则将激光功率设置为 ~ 0.7 kW/cm 2 。通过获得至少3万帧, 在荧光模式下记录原始图像. 
	注: 所有这些参数可以根据不同的试样和标记密度而变化. </li>
	<li> 从原始数据中, 生成一个重建事件列表 (定位在原始图像中闪烁的每个分子), 使用30光子的检测阈值 (需要根据样本进行调整), 方法是单击并 #34; 评估和 #34; 在和 #39; t 系列分析和 #39; #39; 工具和 #39;. </li>
	<li> 按高斯管接头显示超分辨率图像 16 通过单击和 #34 应用渲染像素大小; 创建图像和 #34; 在和 #39 中; 在和 #39; #39; 工具. #39; 
	注: 为了生成超分辨图像, 本研究采用了系统集成软件工具。但是, 所描述的超分辨率成像可以在任何其他单分子定位系统上执行, 并且可以使用开放源码软件工具处理数据, 如雷雨 17 . </li>
</ol> 4。白金阴影 <ol> <li> 移除硅片条纹并在晶片边缘附近添加一滴 PBS, 将其从培养皿中抬起。在 pbs 中清洗硅片 2x 2 分钟, 然后在 pbs 中用0.1% 戊二醛进行修复, 5 分钟洗涤 2x 2 分钟/洗涤. </li>
	<li> 在冰上的水中, 孵育5分钟/孵化1滴纤维素2% 的晶圆片两次。在离心管中插入硅片, 在 14100 x g 的九十年代离心机. 将其安装在扫描电镜下的铝制存根上, 导电碳水泥. </li>
	<li> 在样品上添加一层2到 10 nm 的铂/碳, 旋转阴影在8和 #176; 使用电子束蒸发装置设置: 1.55 伏, 55 毫安, 0.3 nm/秒, 角度8和 #176;, 旋转级别 4. </li>
</ol> 5。扫描电子显微镜 <ol> <li> 具有扫描电子 m 的图像剖面icroscope 在1.5 伏, 2 毫米工作距离和在透镜二次电子探测器. </li>
</ol> 6。光和电子显微镜的对准图像 <ol> <li> 通过单击和 #34 打开两种类型的图像, 并通过斐济 18 ; 文件 |开放和 #34;。通过单击和 #34 调整画布大小; 图像 |调整 |画布大小和 #34; 通过单击和 #34 将两个图像带到堆栈中; 图像 |栈 |图像堆叠和 #34;。将堆栈另存为 tiff 文件类型. </li>
	<li> 在斐济, 通过单击和 #34 打开一个新的 TrackEM2 19 接口; 文件 | 新建 |TrackEM2 (新) 和 #34;。通过右键单击黑窗口并选择和 #34 导入堆栈和两个图像; 导入栈和 #34;. </li>
	<li> 使用鼠标右键单击图像并选择和 #34, 将光学显微图像与电子显微镜图像手动对齐。手动将图层与地标和 #34 对齐;。
	<ol> <li> 选取 "选择" 工具 (黑色箭头) 以添加地标。使用核的形状作为参考, 以在两个图像中选择相同的边 ( 补充图 1 )。添加几个点 (至少需要选择三点)。使用鼠标右键单击仿射模型, 并选择和 #34; 应用转换 |仿射模型与 #34;. </li>
	</ol> </li> <li> 更改图层透明度 (参见 补充图 1 ) 以评估对齐的质量. </li> </ol>

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作者没有什么可透露的。

该方法结合构蛋白定位与上下文信息, 以确定蛋白质在一个细胞中的精确位置。我们在这里展示了实验的完成, 以可视化 Tom20 在线粒体外膜中的表达, 以及它与其他细胞器的关系, 比如在斑马鱼视网膜幼虫中的细胞核或感光细胞的外段。
得冷冻切片需要一些训练, 以获得保存完好的部分。但是, 这是在许多实验室中使用的方法, 并显示成功21。部分的转移到硅片是非常简单的, 不需要特别考虑。使用甘油在成像缓冲是一个非常关键的步骤, 以避免干燥的部分。对于超分辨率成像, 当晶片与培养皿的玻璃底部非常接近时, 获得最佳结果。硅胶条纹有助于保持晶片在这个位置。在去除条纹时要特别小心, 以避免损坏节。
冰层的厚度, 大约 100 nm, 比 Z 维度中的光学分辨率更薄, 此外, 由于超分辨率显微镜信号仅从这个薄薄的图层和扫描电子显微镜信号显示样品的形貌。这种方法也可以与多色成像相结合。然而, 必须特别小心, 样品可以在相同的条件下成像 (如图像缓冲) 和交叉通话应防止。
该方法的一个局限性是它的二维方法, 因为只有有限数量的串行部分 (大约3到7每个晶圆) 可以收集。因此, 处理体积分析的项目并不理想。然而, 它是一种方法, 可用于检测蛋白表达的任何类型的组织, 通过简单的切片样品。我们提供的方法不使用经典的造影剂, 如醋酸铀或柠檬酸铅。白金阴影的对比提供了非常翔实的地形对比, 但在某些情况下, 膜很难解决。这可能会给项目带来问题, 例如, 需要确定小泡中蛋白质的表达。
我们的协议, 基于得冰冻切片, 使用标准设备, 以获得超分辨率和扫描电子显微镜的相关结果。收集硅片上的切片和使用铂的对比是简单的步骤, 提供稳定性和重现性的样品制备。

方法, 以确定蛋白质的亚细胞定位和它们之间的关系, 以不同的单元, 是了解它们的功能和可能的相互作用的基本工具。超分辨率显微镜结合电子显微镜提供了这样的信息1。基态损耗显微镜后, 个别分子返回 (GSDIM) 是一个超分辨率显微镜技术兼容广泛的有机和遗传编码的荧光2和实现横向分辨率高达 20 nm3. 采用比标准衍射极限显微镜更高分辨率的方法, 提高了相关性4、56的准确性。为了达到与特定亚细胞室的蛋白质表达的最佳相关性, 并减少不确定度的体积7推荐使用相同的超薄切片进行光和电子显微镜。在不同的切片方法中, 得冰冻切片协议不需要脱水或树脂嵌入, 此外, 保留了许多表位抗原性, 并提供了良好的组织超微结构8。几种方法在相关光和电子显微镜 (克莱)4、5、9、10中显示了这些部分的适用性。
斑马鱼视网膜是一个有价值的模型, 研究视觉发展和人类疾病的机制, 鉴于其高度保守的结构和功能, 在脊椎动物。特别是, 视网膜光显示与哺乳动物光相同的结构, 与基底突触, apico 基部拉长核, 线粒体聚类在更顶端内段和外段组成的膜最顶端位置的磁盘11。蛋白质定位到不同的细胞隔间是保存在斑马鱼和人类之间, 允许调查的生物学功能的人类疾病相关的蛋白质12,13。
在这里, 我们提出了一个协议来准备斑马鱼视网膜标本, 以解决线粒体外膜蛋白 Tom20 的相关超分辨光和电子显微镜的定位。该方法是基于在硅片上收集低温切片, 并通过在应用了一层铂后产生的地形信息获得对比。这些步骤在易用性、重现性和完成实验的时间方面都有明显的技术改进。我们最近证明了该方法在小鼠组织中检测核孔和线粒体蛋白的适用性14。

<table>
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			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>#158127</td>
			<td></td>
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			<td>Glutaraldehyde EM Grade</td>
			<td>EMS, USA</td>
			<td>#16220</td>
			<td></td>
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			<td>Cacodylate</td>
			<td>Merck</td>
			<td>#8.20670</td>
			<td></td>
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			<td>Tricaine</td>
			<td>Sigma-Aldrich</td>
			<td>#886-86-2</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose, peqGOLD Universal</td>
			<td>VWR International GmbH</td>
			<td>35-1020</td>
			<td></td>
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			<td>Flat embedding molds</td>
			<td>BEEM Flat</td>
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			<td>Local food brand gelatin</td>
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			<td>Merck</td>
			<td>#1.07687</td>
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			<td>Methylcellulose</td>
			<td>Sigma</td>
			<td>#M-6385</td>
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			<td>Sigma</td>
			<td>#G-7126</td>
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			<td>Gelatin type B</td>
			<td>Sigma</td>
			<td>#G-6650</td>
			<td></td>
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			<td>BSA</td>
			<td>Applichem</td>
			<td>#A6588.0050</td>
			<td></td>
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			<td>Silicon wafer</td>
			<td>Si-Mat Silicon Materials</td>
			<td></td>
			<td>Type: P/Boron; Orientation &#60;111&#62; ON; Growth method: CZ; Resistivity:1-30 ohm/cm; Surface: polished; Laser cut at 7 x7 mm</td>
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		<tr>
			<td>Cryo-pin</td>
			<td>Baltic Preparation</td>
			<td>#16701950</td>
			<td></td>
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			<td>Wired loop &#34;Perfect loop&#34;</td>
			<td></td>
			<td></td>
			<td></td>
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		<tr>
			<td>Silicone stripes</td>
			<td>Picodent</td>
			<td>Twinsil 22</td>
			<td></td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Sigma-Aldrich</td>
			<td>#G8270</td>
			<td></td>
		</tr>
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			<td>glucoseoxidase</td>
			<td>Sigma-Aldrich</td>
			<td>#G7141</td>
			<td></td>
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			<td>catalase</td>
			<td>Sigma-Aldrich</td>
			<td>#C40</td>
			<td></td>
		</tr>
		<tr>
			<td>beta-Mercaptoethylamine hydrochloride</td>
			<td>Sigma-Aldrich</td>
			<td>#M6500</td>
			<td></td>
		</tr>
		<tr>
			<td>anti-Tom20</td>
			<td>Santa Cruz Biotechnology</td>
			<td>#sc - 11415</td>
			<td></td>
		</tr>
		<tr>
			<td>AlexaFluor 647 AffiniPure F(ab&#39;)2 fragment donkey anti rabbit IgG</td>
			<td>Jackson Immuno-Research</td>
			<td>#711-606-152</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>ROCHE, Switzerland</td>
			<td>#10236276001</td>
			<td></td>
		</tr>
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			<td>Glycerol solution</td>
			<td>Sigma-Aldrich</td>
			<td>#49782</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass bottom petri dish</td>
			<td>Ibidi, Germany</td>
			<td>u-Dish 35mm, high glass bottom, #81158</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM aluminium stub</td>
			<td>Agar Scientific</td>
			<td>#G301F</td>
			<td></td>
		</tr>
		<tr>
			<td>Conducting carbon cement Leit-C</td>
			<td>Plano, Germany</td>
			<td>AG3300</td>
			<td></td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond Knife (Cryo Immuno)</td>
			<td>Diatome</td>
			<td>DCIMM3520</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryo-ultramicrotome</td>
			<td>Leica Microsystems</td>
			<td>Leica EM FCS</td>
			<td></td>
		</tr>
		<tr>
			<td>Widefield-TIRF microscope GSD Leica SR GSD 3D</td>
			<td>Leica Microsystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>160x 1.43 TIRF objective</td>
			<td>Leica Microsystems</td>
			<td>11523048</td>
			<td></td>
		</tr>
		<tr>
			<td>SEM - Zeiss Supra 50 VP</td>
			<td>Zeiss</td>
			<td>Supra 50 VP</td>
			<td></td>
		</tr>
		<tr>
			<td>FIB-SEM - Zeiss Auriga 40</td>
			<td>Zeiss</td>
			<td>Auriga 40</td>
			<td></td>
		</tr>
	</tbody>
</table>

correlative super-resolution and electron microscopy to resolve protein localization in zebrafish retina

本文采用超分辨光学显微镜和扫描电镜相结合的方法来研究斑马鱼视网膜的亚细胞蛋白定位。超分辨率光显微镜的 sub-diffraction 极限分辨能力可以提高相关数据的准确性。简要地, 110 纳米厚的低温切片转移到一个硅片, 并在免疫荧光染色后, 图像的超分辨率光显微镜。随后, 在扫描电子显微镜 (SEM) 成像之前, 这些切片被保存在纤维素和铂阴影中。从这两个显微镜模式的图像很容易合并使用开放源码软件的组织地标。在这里, 我们描述了适应方法的幼虫斑马鱼视网膜。然而, 这种方法也适用于其他类型的组织和有机体。我们证明, 这种相关性获得的补充信息能够解决线粒体蛋白的表达与线粒体膜和嵴的关系, 以及细胞的其他舱室。

蛋白 Tom20 的表达, translocase 线粒体外膜复合体的亚基20, 是确定的, 在薄切片的斑马鱼视网膜, 通过超分辨率光显微镜 (图 1), 这一信息是补充与扫描电子显微镜获得的地形信号后, 铂阴影相同的部分。这些相关数据证实了蛋白质与某一特定的隔间、外线粒体膜的结合, 另外还提供了有关蛋白质与细胞的其他器官器的关系的信息。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56113/56113fig1.jpg"> 
图 1: 在斑马鱼视网膜上的克莱克.a.低放大率 widefield 图像 5 dpf 斑马鱼视网膜切片, 细胞核染色 DAPI (青色)。B.同一区域的扫描电子显微镜。C.高放大倍数 widefield 图像的帧在 b. DAPI (青色) 和 Tom20 线粒体染色的细胞核呈红色。D.在更高的放大倍数下 Widefield 相同剖面的图像。Tom20 表达的模式是线粒体的聚集。E. GSDIM 显微镜检测到的 Tom20 (红点) 的表达。核染色的 DAPI (青色)。F.与相关超分辨率和扫描电子显微镜相结合的 E 节。线粒体外膜上的线粒体簇 (M) 出现 Tom20 染色 (红点)。荧光 DAPI 信号在原子核 (N) 对应与 SEM 图象的地势。G. F 帧的高放大图像。扫描电子显微镜图像提供了 GSDIM 图像 (红点) 的上下文。线粒体嵴明显可见, Tom20 染色被定位于线粒体外膜。外段膜的光 (OS) 是明确解决。图像像素大小 5 nm。刻度栏: A、B 和 C:10 µm;D: 2 µm;E 和 F: 1 µm 和 G: 0.2 µm.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56113/56113fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
补充图 1: 光和电子显微图像的对准。TrackEM2 接口的. 屏幕截图, 带有 SEM 图像和不同核的编号地标 (黄色)。B. TrackEM2 界面上的屏幕截图与荧光图像和编号地标 (黄色) 沿不同 DAPI 染色核。要更改图层透明度, 可以使用菜单左上部分的滑块。缩放栏: 1 µm.<a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56113/Supplementary_Figure_1.tif">请单击此处下载此文件.</a>

Watch this Scientific Journal Video about Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina at JoVE.com

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

该协议描述了在斑马鱼视网膜上获得亚细胞蛋白定位结果的必要步骤, 方法是将超分辨光显微镜和扫描电子显微镜图像关联起来。

斑马鱼视网膜蛋白定位的相关超分辨和电镜观察

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

correlative-super-resolution-electron-microscopy-to-resolve-protein

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

9.4K 次观看.  University of Zurich. 该协议描述了在斑马鱼视网膜上获得亚细胞蛋白定位结果的必要步骤, 方法是将超分辨光显微镜和扫描电子显微镜图像关联起来。

视频: Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

测量生活中的斑马鱼胚胎蛋白质稳定性使用荧光衰减光转化后（FDAP）

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

科研

发育生物学

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 ...

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain&#39;s macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

健康和受伤斑马鱼脑的大型扫描透射电子显微镜（Nanotomy）

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

配备了光学切片与成像定时使用解剖显微镜斑马鱼肾脏发展分析

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Using Light Sheet Fluorescence Microscopy to Image Zebrafish Eye Development

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM compared to confocal systems are its low phototoxicity, gentle mounting strategies, fast acquisition with high signal to noise ratio and the possibility of imaging samples from various angles (views) for long periods of time. Imaging from multiple views unleashes the full potential of LSFM, but at the same time it can create terabyte-sized datasets. Processing such datasets is the biggest challenge of using LSFM. In this protocol we outline some solutions to this problem. Until recently, LSFM was mostly performed in laboratories that had the expertise to build and operate their own light sheet microscopes. However, in the last three years several commercial implementations of LSFM became available, which are multipurpose and easy to use for any developmental biologist. This article is primarily directed to those researchers, who are not LSFM technology developers, but want to employ LSFM as a tool to answer specific developmental biology questions. 
Here, we use imaging of zebrafish eye development as an example to introduce the reader to LSFM technology and we demonstrate applications of LSFM across multiple spatial and temporal scales. This article describes a complete experimental protocol starting with the mounting of zebrafish embryos for LSFM. We then outline the options for imaging using the commercially available light sheet microscope. Importantly, we also explain a pipeline for subsequent registration and fusion of multiview datasets using an open source solution implemented as a Fiji plugin. While this protocol focuses on imaging the developing zebrafish eye and processing data from a particular imaging setup, most of the insights and troubleshooting suggestions presented here are of general use and the protocol can be adapted to a variety of light sheet microscopy experiments.

Light sheet fluorescence microscopy is an excellent tool for imaging embryonic development. It allows recording of long time-lapse movies of live embryos in near physiological conditions. We demonstrate its application for imaging zebrafish eye development across wide spatio-temporal scales and present a pipeline for fusion and deconvolution of multiview datasets.

采用轻质板材荧光显微镜到图像斑马鱼眼睛发育

Light sheet fluorescence microscopy (LSFM) is gaining more and more popularity as a method to image embryonic development. The main advantages of LSFM ...

Analysis of Protein-protein Interactions and Co-localization Between Components of Gap, Tight, and Adherens Junctions in Murine Mammary Glands

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These interactions are provided by junctional proteins that form nexuses between adjacent cells. Junctional protein mislocalization and reduced physical associations with their binding partners can result in the loss of function and, consequently, to organ dysfunction. Thus, identifying protein localization and protein-protein interactions (PPIs) in normal and disease-related tissues is essential to finding new evidences and mechanisms leading to the progression of diseases or alterations in developmental status. This manuscript presents a two-step method to evaluate PPIs in murine mammary glands. In protocol section 1, a method to perform co-immunofluorescence (co-IF) using antibodies raised against the proteins of interest, followed by secondary antibodies labeled with fluorochromes, is described. Although co-IF allows for the demonstration of the proximity of the proteins, it does make it possible to study their physical interactions. Therefore, a detailed protocol for co-immunoprecipitation (co-IP) is provided in protocol section 2. This method is used to determine the physical interactions between proteins, without confirming whether these interactions are direct or indirect. In the last few years, co-IF and co-IP techniques have demonstrated that certain components of intercellular junctions co-localize and interact together, creating stage-dependent junctional nexuses that vary during mammary gland development.

Intercellular junctions are requisites for mammary gland stage-specific functions and development. This manuscript provides a detailed protocol for the study of protein-protein interactions (PPIs) and co-localization using murine mammary glands. These techniques allow for the investigation of the dynamics of the physical association between intercellular junctions at different developmental stages.

蛋白质 - 蛋白质相互作用的分析和鼠乳腺中间隙，紧密和粘附连接部位之间的共定位

Cell-cell interactions play a pivotal role in preserving tissue integrity and the barrier between the different compartments of the mammary gland. These ...

Multimodal Hierarchical Imaging of Serial Sections for Finding Specific Cellular Targets within Large Volumes

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a combination of light and electron microscopy. Samples embedded in resin are sectioned into arrays consisting of ribbons of hundreds of ultrathin sections and deposited on pieces of silicon wafer or conductively coated coverslips. Arrays are imaged at low resolution using a digital consumer like smartphone camera or light microscope (LM) for a rapid large area overview, or a wide field fluorescence microscope (fluorescence light microscopy (FLM)) after labeling with fluorophores. After post-staining with heavy metals, arrays are imaged in a scanning electron microscope (SEM). Selection of targets is possible from 3D reconstructions generated by FLM or from 3D reconstructions made from the SEM image stacks at intermediate resolution if no fluorescent markers are available. For ultrastructural analysis, selected targets are finally recorded in the SEM at high-resolution (a few nanometer image pixels). A ribbon-handling tool that can be retrofitted to any ultramicrotome is demonstrated. It helps with array production and substrate removal from the sectioning knife boat. A software platform that allows automated imaging of arrays in the SEM is discussed. Compared to other methods generating large volume EM data, such as serial block-face SEM (SBF-SEM) or focused ion beam SEM (FIB-SEM), this approach has two major advantages: (1) The resin-embedded sample is conserved, albeit in a sliced-up version. It can be stained in different ways and imaged with different resolutions. (2) As the sections can be post-stained, it is not necessary to use samples strongly block-stained with heavy metals to introduce contrast for SEM imaging or render the tissue blocks conductive. This makes the method applicable to a wide variety of materials and biological questions. Particularly prefixed materials e.g., from biopsy banks and pathology labs, can directly be embedded and reconstructed in 3D.

This protocol targets specific cells in tissue for imaging at nanoscale resolution using a scanning electron microscope (SEM). Large numbers of serial sections from resin-embedded biological material are first imaged in a light microscope to identify the target and then in a hierarchical manner in the SEM.

用于在大容量中查找特定细胞目标的串行剖面的多模式分层成像

Targeting specific cells at ultrastructural resolution within a mixed cell population or a tissue can be achieved by hierarchical imaging using a ...

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 ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

全斑马鱼胚胎的延长时移共聚焦显微镜的分层安装方法

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...