作者感谢过去和现在我们实验室的成员谁帮助开发这些技术。由来自美国加州理工学院的再生医疗/ CIRM（RN1-00538）的资助提供给DE部分资助。

1，斑马鱼饲养，胚胎发生和治疗<ol><li>进行机构动物护理和使用委员会（IACUC）的指导下，并在美国国立卫生研究院或其他监管机构/指引的动物护理指引下斑马鱼的协议。 </li><li>可从国际斑马鱼资源中心（ZIRC）表达的荧光蛋白在特定组织，细胞或器官的斑马鱼品系。例如，玻璃转化温度（KDR：EGPF）S843中血管内皮细胞6，它可以被用来产生完整的三维血管结构如本协议表达EGFP。可从ZIRC其它转基因斑马鱼。 </li><li>房子成年斑马鱼在一个合适的养殖系统，可以监测pH值，盐度，温度，溶解氧，光照等环境因素7。这里显示的斑马鱼被安置在一个Aquaneering公司系统（圣地亚哥，加利福尼亚州）在28.5℃下用14小时LIGht/10小时黑暗循环。喂成年斑马鱼丰年虾和NRD 4/6鱼饲料（丰年虾直接，奥格登，犹他州）的均衡饮食。 </li><li>成年男性和女性的斑马鱼繁殖的年龄应分别容纳增加交配成功。 </li><li>刺激产卵和受精放置女性（2-4）和男性（4-6）一起在具有网状底部有孔足够大的鸡蛋落在通过交配容器，但太小的斑马鱼成人传递。设置在交配前一天晚上;产卵后不久的黎明，而平时白天光照周期缓慢增加强度（黎明）。检查鸡蛋在嵌合容器的底部每隔15-30分钟。 </li><li>使用网状过滤器和干净的E3缓冲液（5 mM氯化钠，0.17毫米氯化钾，0.33毫米氯化钙 ，0.33毫米硫酸镁4）收集鸡蛋。转蛋到100mm培养皿在28℃培养箱中充满了E3的缓冲和存储。 </li><li>研究认为改变CER化学品ebrovascular分支添加所需的化学浓度。例如neovasular分支可诱发与γ-分泌酶抑制剂（GSI IX/DAPT/N- [N-（3,5 - Difluorophenacetyl-L-丙氨酰基）]-S-phenylglycinet丁基酯）溶解在DMSO 4，开始24小时后受精（HPF）。在那个时间点的胚胎仍然是绒毛膜内，许多化学物质可以通过它8。如果一个处理条件有神经或马达效应可能损害胚胎挣脱绒毛膜的能力，那么胚胎应取消chorionated 24和​​48个高倍视野，可以用链霉蛋白酶2的一方钳子来完成的。在所提供的图像显示胚胎轻轻抓住了两个削尖钳绒毛膜和撕裂它打开dechorionated。 </li><li>如果需要/想要的，色素形成，可以通过添加0.003％的N-苯基硫脲嘧啶（PTU）的E3缓冲区24 HPF抑制。 </li></ol> 2。共焦脑结构的成像在固定的斑马鱼胚胎URES <ol><li>牺牲在250 mg / L的三卡因甲磺酸盐的胚胎，然后修复它们通过浸没在2 - 4％多聚甲醛固定过夜，在4℃下容器荧光的胚胎应该用铝箔包裹。一旦固定，胚胎应贮存在PBS中于4℃直至成像 - EGFP的荧光强度变小解决后一个星期左右，但形态（如在亮视野观察）被更长的保留。 </li><li>准备通过先用锋利的钨针取出一只眼睛来装载胚胎。围绕连接眼部组织切割第一再切肌肉，并最终切断视神经置换的眼睛。 （注：如果成像双方需要删除这两个眼睛。） </li><li>一旦眼睛被删除，安装胚上用降3％甲基纤维素的玻璃罩。定向的胚胎，使与被删除的眼睛的侧面向盖玻片，并尽可能靠近玻璃越好。盖上甲基整个胚胎lcellulose以防止成像过程中干燥。 </li><li>图像立即使用倒置共聚焦显微镜配备有高品质的20倍计划的Apo物镜（数值孔径为0.75或更高）的安装胚胎。这种配置是优选的非倒置显微镜，将需要夹着两个玻璃面之间的胚胎，并按下胚胎靠在顶玻璃。 </li><li>收集光切片1微米使用中等或大光圈设定单位。也可以使用较大的步长为2.5微米，但它可能会更加困难，以确定较小的对象的空间的顺序。小孔可产生更清晰的细节，但需要较长的扫描可漂白EGFP和也限制成像的深度成可达到的胚胎。共聚焦显微镜，可以对成像大约一半的方式，通过一个胚胎。 </li><li>如果整个鱼的成像需要，在一开始就取出两只眼睛（见注在步骤3.2），IMAGIN后旋转胚胎克1侧，并重复步骤2.3 - 2.5的相对侧上。 </li></ol> 3。3D胚胎斑马鱼Cerebrovasculature重建<ol><li>使用开源的ImageJ的斐济分布3（http://fiji.sc），这是用于3D渲染的优化，完全免费，并与PC，Mac和Linux计算机兼容。 </li><li>进口焦堆到斐济前往插件&gt; LOCI&gt; BioFormats进口商9然后选择共焦文件， 例如 name.ids尼康栈（ 图2A）。选择与Hyperstack，彩色模式查看堆栈：灰度，自动定标检查，检查分裂渠道。 </li><li>这些选项会打开四个独立的通道板;对于EGFP只有一个会是必要的，通常是第三个了。关闭其他三个面板（红色，蓝色和alpha）留下16位图像具有长名称后跟C = 1（ 图2A）。 </li><li>虽然图像使用滚动TA通过扫描调整门槛B随着底部找到一个切片，有感兴趣的区域或结构，然后去图像&gt;调整&gt;阈值（ 图2C）。 </li><li>在出现的面板，滑动顶部栏（黑电平）到左边，这样的结构可以看出，相当不错了背景，并留下底部的幻灯片（白电平），它在哪里（ 图2D）。选择B＆W的，选择深色背景。不要选择阈值计算为每个图像，除非需要不同的调整为每个切片，选择黑色的背景。此过程将创建一个新的8位图像。 </li><li>警告：阈值不能撤销或保存在ImageJ的，所以共焦堆栈将有一个改变是必要每次都重新加载。写下数字，并尝试不同的设置，直到想要的效果。 </li><li>转到插件&gt; 3D查看器&gt;阈值0，重采样因子1（最好）或2（好），取消选择红色和蓝色方块，以绿色3D输出 - 否则无线LL产生白色渲染-选择Apply（不自动）（ 图2E）。 </li><li>旋转，旋转，并使用鼠标和键盘控制（ 图2F）放大的三维图像。 </li><li>通过使用捕捉选项菜单中保存静止图像的任何一点。屏幕上的图像框的大小决定了图像的生成像素尺寸，所以如果一个高分辨率图像需要使框拖动右下角较大。 </li><li>通过选择View&gt;录制360度旋转（ 图2G）创建一个旋转的3D电影。旋转默认为每步2度，但是它可以改变为例如5度，这将造成更小的文件大小，但也可以添加到急动的动画。 </li><li>将文件保存在几个可用的格式之一后用媒体播放器观看，上传到互联网，或使用PowerPoint演示文稿。开源媒体播放器，VLC（ <a href="http://www.videolan.org/vlc/index.html">HT</a>TP :/ / www.videolan.org / VLC / index.html）上，是免费的，并处理这些视频非常好。 </li></ol>

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没有什么可以透露。

这里所描述的方法提供了血管系统的开发视觉斑马鱼的研究奠定了基础。活体标本可以被用来评估生理参数，如心脏速率和心脏每搏输出量，而固定的样品可用于高分辨率共焦成像。 果蝇和C。线虫允许全身显像，但斑马鱼是脊椎动物，并提供脊椎动物组织中一个有用的模型，包括内皮细胞衬里的血管系统。这些研究可以从斑马鱼研究领域包括显著转基因株系和基因组资源。三维重建和共聚焦图像从斑马鱼胚胎的渲染，这里描述的，允许一个系统生物学方法血管分支和血管密度是不可能的更大的动物模型如大鼠和小鼠。此外，由于羊膜斑马鱼开发一个可修改的环境（E3缓冲区），在那里人们可以很容易地将chemicals抑制，​​影响血管发育特异性酶或其他进程。化学品输送的浓度和时间可以改变，从而使研究者能够微调处理条件。 1，修改和故障排除修改本系统可以结合斑马鱼的转基因株系中表达的EGFP或其它荧光蛋白在多种组织，器官或区域的特定图案10。此外，在斑马鱼视网膜新生血管的变化分析已出版5。与色素沉着的老年胚胎和成年斑马鱼的问题可以通过用不产生规模颜料或视网膜色素转基因线交叉进行补偿。用荧光减弱的问题通常不恰当的固定条件的结果。多聚甲醛（4％）1天是最佳的，但更强的固定剂，如戊二醛，锇酸或醇，可摧毁绿色荧光蛋白的荧光。固定后，胚胎应保持在PBS中于4℃下，总是避免阳光直射。 这种技术2。限制与此协议制作的3D效果图的质量和分辨率取决于生成的图像的质量。通过这些胚胎光线穿透使用标准的共聚焦显微镜限定于正中矢状平面。成像的该方面限制了成像的深度在老年人胚胎和成人，但是更高级的多光子显微镜系统允许成像在更大的深度。 3，相对于现有方法的意义该协议提供了一种方法，血管网络上的系统级，可以将整个动物的分析。这些数据的早期交涉往往依赖于一系列布局在一起的图像，但3D渲染提供更好的分辨率的空间关系的tionships参与。 4，未来的应用在成像和组织处理的新发展将提供许多新的应用，这些方法可能包括让年龄较大的胚胎或成人透明11-14。增强透明度，通过共聚焦和多光子激光大大增加组织渗透。此外，由于相机和光电倍增管的速度增加它可能很快就可以生产鱼的3D渲染实时，提供分析的4 个维度。 5，关键步骤在这个协议的一个关键步骤是准备成像，其中包括适当的固定。成像应固定与可得的最佳数值孔径高品质的目标后尽快完成。分辨率取决于所使用的成像系统上，所以高品质的系统通常会更好。产生3D效果图是内存密集型为使新的，高端计算机与大量的内存和良好的图形处理器推荐。 这里所描述的视觉生物学系统已被优化，表达EGFP的血管内皮细胞的转基因斑马鱼，虽然这些方法可适于表达绿色荧光蛋白，或其它荧光蛋白，在神经元，肌肉，腺体或任何数目的种群的转基因胚胎其他细胞。在这个系统工作的主要优点是研究什么是发生在整个胚胎在任何时间在此发展时期，在固定和/或活的动物的能力。

斑马鱼提供了强大的系统来研究发育生物学，及其胚胎的相对透明度是服从，影像为基础的研究1。斑马鱼已被用来作为一种模式脊椎动物发展了几十年。硬骨鱼类，包括斑马鱼，有一个简单的脊椎动物血管系统，有没有合理的同源基因在脊椎动物。血液从一个两腔心脏前房通过鳃，在那里它被氧化的泵送。从鳃血液会聚在背主动脉和穿过动脉分支成更小和更小的血管，最终到达毛细血管在器官组织中。内毛细血管氧释放和二氧化碳被吸收。上毛细血管的静​​脉侧血液流入越来越大静脉和最终被吸入到心脏，其中，循环重复的后房。 成年斑马鱼可产卵200以上鸡蛋的时候，和ONCË受精，它们很快地发展2。在一天之内身体纵轴非常发达，包括肌肉收缩和移动的胚胎绒毛膜内。从2-7天受精后（DPF）最体系统开发，包括眼睛和中枢神经系统，可以协调对食物或远离强光游泳。多达7旦胚胎是足够小，以允许可视化用显微镜。表达荧光蛋白的转基因株系可与成像或共聚焦荧光显微镜。共焦成像可搭配开源软件3在斑马鱼胚胎，提供血管发育的系统生物学的角度建立完整的血管结构的三维效果图。研究涉及改变血管和脑血管的复杂性将受益于这一协议，因为它允许血管网络4,5的系统级分析。的方法和资源的汇编是提供d来允许这些技术很容易通过和需要血管结构在胚胎斑马鱼的影像学检查。斑马鱼作为动物模型的成本效率相结合的新兴的成像技术，提供与评估在脊椎动物的发育和稳态的分子途径的血管生成作用的新平台。

<table border="0" cellpadding="0" cellspacing="0" width="889">
	<tbody>
		<tr>
			<td>N &#8211; Phenylthiourea&#160;</td>
			<td>Alfa Aesar, catalog #41972</td>
			<td></td>
			<td>0.2 M in E3 buffer, kept at 4oC</td>
		</tr>
		<tr>
			<td>E3 buffer</td>
			<td>Sigma</td>
			<td></td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4</td>
		</tr>
		<tr>
			<td>Confocal microscope&#160;</td>
			<td>Nikon</td>
			<td></td>
			<td>D-EclipseC1 on a Nikon TE-2000U</td>
		</tr>
		<tr>
			<td>Glass bottom dishes</td>
			<td>Mat-Tek</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GSI IX/DAPT</td>
			<td></td>
			<td></td>
			<td>N-[N-(3,5-Difluorophenacetyl-L-alanyl)]-S-phenylglycinet-butyl ester EMD Biosciences</td>
		</tr>
		<tr>
			<td>24 well plates&#160;</td>
			<td>Becton-Dickinson, cat# 351147</td>
			<td></td>
			<td>BD Falcon</td>
		</tr>
		<tr>
			<td>Transfer pipettes&#160;</td>
			<td>VWR,&#160; cat #414004-001</td>
			<td></td>
			<td>VWR disposable transfer pipets</td>
		</tr>
		<tr>
			<td>Methyl-cellulose&#160;</td>
			<td>Alfa Aesar, cat#43146</td>
			<td></td>
			<td>3% in E3 buffer</td>
		</tr>
		<tr>
			<td>NRD 4/6 Fish food&#160;</td>
			<td>Brine Shrimp Direct</td>
			<td></td>
			<td>Dried</td>
		</tr>
		<tr>
			<td>Brine shrimp</td>
			<td>Brine Shrimp Direct</td>
			<td></td>
			<td>Live</td>
		</tr>
		<tr>
			<td>Tungsten wire&#160;</td>
			<td>Small Parts # TW-016-60</td>
			<td></td>
			<td>0.016&#8221; OD</td>
		</tr>
		<tr>
			<td>Tricaine</td>
			<td>VWR # 101107-950</td>
			<td></td>
			<td>Tricaine methanesulfonate 250 mg/L in E3 buffer</td>
		</tr>
	</tbody>
</table>

imaging and 3d reconstruction of cerebrovascular structures in embryonic zebrafish

斑马鱼是一种强大的工具来研究发育生物学和病理学的体内 。小尺寸和斑马鱼胚胎的相对透明度使其成为如心脏和血管发育过程中的目视检查特别有用。在最近的一些研究，表达EGFP的血管内皮细胞中的转基因斑马鱼被用于图像和分析复杂的血管网络在大脑和视网膜，使用共聚焦显微镜。提供说明的准备，处理和图像表达增强型绿色荧光蛋白（EGFP）斑马鱼胚胎，然后生成脑血管系统的全面的3D效果图。协议包括治疗胚胎，共焦成像，和固定通讯协定，保护绿色荧光蛋白的荧光。此外，设置在获取脑结构，如摘除眼球不破坏附近的神经组织的高品质图像的有用提示。潜在的陷阱用共聚焦成像进行了讨论，同时要生成共聚焦图像堆栈使用免费的开源软件，三维重建的步骤。

三维重建血管结构提供了斑马鱼发育的全面和有趣的视觉透视图1和图2示出的方法，因为它们典型地完成。 图3示出的血管结构中有6单丝旦的斑马鱼胚胎所表达EGFP的在内皮细胞中的多个角度。有了坚实的绿色或白色可能很难体会到的信号强度;伪彩色环从一个查找表提供的图像强度，并且允许更好的深度知觉时结构重叠。在脉管系统中的伪色的三维图像的一个例子的6单丝旦的斑马鱼在图4中提供。活胚胎的荧光成像可以被用来研究生理特征，包括眼睛和身体的运动，和心脏活动。 图3和图4显示用这些方法得到的典型结果，利用转基因斑马鱼线<html>描述。成像分辨率取决于显微镜的特点，但EGFP信号的亮度足以满足大多数商业系统良好的图像质量。重建的三维表示和渲染是一致的，这个开源软件中选择提供始终如一的好成绩。 <img alt="图1" fo:content-width="6in" src="/files/ftp_upload/50417/50417fig1highres.jpg" width="600" /> 图1。眼睛的去除。 a）固定 3 DPF胚胎与钨需求定位旁边的眼球。组织从这个位置。 乙切眼部周围）的眼睛是脱落和底层眼部肌肉和视神经被切断。空眼窝表示用虚线圆圈。C）相同的胚胎被翻转并安装有甲基纤维素，与完整的眼朝上。<html> hres.jpg“目标=”_blank“&gt;请点击这里查看该图的放大版本。 <img alt="图2" fo:content-width="6in" src="/files/ftp_upload/50417/50417fig2highres.jpg" width="600" /> 图2步骤一步的共聚焦图像堆栈的三维重建。 A）使用插件&gt; LOCI&gt; Bioformat选择B）找到一个切片与感兴趣区域内后，斐济加载打开文件（4104.1.ids），阈值调整被选中，如图所示。C）阈值是使用顶部调整到214滑块和应用被选中D）3D查看器被称为如图E）的三维重建显示用眼完好斑马鱼，定向F）的图像已被缩放和旋转。G）360度旋转的电影制成，如图所示。<html> res.jpg“目标=”_blank“&gt;请点击这里查看该图的放大版本。 <img alt="图3" fo:content-width="6in" src="/files/ftp_upload/50417/50417fig3highres.jpg" width="600" /> 图3：从三维重建的观点。一）6 DPF胚胎成像与10倍的目标内侧的角度看，口在右边，嘴巴里面没有鳃，B中的同一胚胎的）侧，注意散热片是在中间的循环。 三）同胚成像与20倍的目标，内侧的角度，注意鳃分辨率D）侧的20倍的目标成像角度。鳍是在面板的右边缘E）安特罗-中层鉴于20倍的目标成像，同样胚胎成像与20倍的目标，它的头向右侧的内口注鳃F）腹部。注意脉管上的卵黄囊在右下角。jove.com/files/ftp_upload/50417/50417fig3highres.jpg“目标=”_blank“&gt;请点击这里查看该图的放大版本。 <img alt="图4" fo:content-width="6in" src="/files/ftp_upload/50417/50417fig4highres.jpg" width="600" /> 用伪彩色查找表为信号强度的6单丝旦重建的6旦胚胎图4。强度的差异。图片。口，脑，鳃和卵黄囊被标记为方向。 <a href="https://www.jove.com/files/ftp_upload/50417/50417fig4highres.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" fo:content-width="5in" src="/files/ftp_upload/50417/Zfish_still.jpg" width="500" /> 重建的血管系统在4 DPF斑马鱼图5。影片。鱼的影像是在2.5微米。该图像是从成像一半的胚胎。比较血管结构与结构中的GSI处理斑马鱼在图6中提供。注意血管在头下的密度和较大的腮。 （参见下载下的“Zfish_spin.avi”补充文件） <img alt="图6" fo:content-width="5in" src="/files/ftp_upload/50417/Zfish_still1.jpg" width="500" /> 图6。血管系统中，在4 DPF一GSI处理胚胎的3D电影。鱼的影像是在2.5微米至从横向到中线。比较血管结构与对照4单丝旦在图5所示的鱼的弓背和更小的尺寸是典型的具有这种化学处理的胚胎。 （参见下载下的“GSI-treated_4dpf_fish.avi”补充文件）

Watch this Scientific Journal Video about Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish at JoVE.com

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

在斑马鱼幼虫脑血管病的发展成像描述。还提供技术，以促进3D成像，并使用化学处理修改脑发展。

成像和胚胎斑马鱼脑血管结构的三维重建

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

imaging-3d-reconstruction-cerebrovascular-structures-embryonic

Research

JoVE Journal

Biology

14.7K 次观看.  Western University of Health Sciences. 在斑马鱼幼虫脑血管病的发展成像描述。还提供技术，以促进3D成像，并使用化学处理修改脑发展。 

视频: Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

现场共聚焦显微镜对斑马鱼的胚胎脑成像

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

科研

生物学

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

组织有针对性的胚胎嵌合体：斑马鱼原肠胚细胞移植

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we present a protocol for the mounting of Drosophila melanogaster embryos and subsequent live imaging of fluorescently labeled hemocytes, the embryonic macrophages of this organism. Using the Gal4-uas system1 we drive the expression of a variety of genetically encoded, fluorescently tagged markers in hemocytes to follow their developmental dispersal throughout the embryo. Following collection of embryos at the desired stage of development, the outer chorion is removed and the embryos are then mounted in halocarbon oil between a hydrophobic, gas-permeable membrane and a glass coverslip for live imaging. In addition to gross migratory parameters such as speed and directionality, higher resolution imaging coupled with the use of fluorescent reporters of F-actin and microtubules can provide more detailed information concerning the dynamics of these cytoskeletal components.

Live Imaging Of Drosophila melanogaster Embryonic Hemocyte Migrations

Drosophila hemocytes disperse over the entirety of the developing embryo. This protocol demonstrates how to mount and image these migrations using embryos with fluorescently labelled hemocytes.

实时成像果蝇胚胎血细胞迁移

Many studies address cell migration using in vitro methods, whereas the physiologically relevant environment is that of the organism itself. Here we ...

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

斑马鱼胚胎眼组织显微切割的

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

免疫染色的解剖斑马鱼胚胎心脏

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

一个系统<em&gt;体外</em培养的胚胎胰腺

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

激光伤害的斑马鱼胚胎骨骼肌

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Analysis of Embryonic and Larval Zebrafish Skeletal Myofibers from Dissociated Preparations

The zebrafish has proven to be a valuable model system for exploring skeletal muscle function and for studying human muscle diseases. Despite the many advantages offered by in vivo analysis of skeletal muscle in the zebrafish, visualizing the complex and finely structured protein milieu responsible for muscle function, especially in whole embryos, can be problematic. This hindrance stems from the small size of zebrafish skeletal muscle (60 &mu;m) and the even smaller size of the sarcomere. Here we describe and demonstrate a simple and rapid method for isolating skeletal myofibers from zebrafish embryos and larvae. We also include protocols that illustrate post preparation techniques useful for analyzing muscle structure and function. Specifically, we detail the subsequent immunocytochemical localization of skeletal muscle proteins and the qualitative analysis of stimulated calcium release via live cell calcium imaging. Overall, this video article provides a straight-forward and efficient method for the isolation and characterization of zebrafish skeletal myofibers, a technique which provides a conduit for myriad subsequent studies of muscle structure and function.

Zebrafish are an emerging system for modeling human disorders of the skeletal muscle. We describe a fast and efficient method to isolate skeletal muscle myofibers from embryonic and larval zebrafish. This method yields a high-density myofiber preparation suitable for study of single skeletal muscle fiber morphology, protein subcellular localization, and muscle physiology.

从解离分析准备胚胎和幼体斑马鱼骨肌纤维

The zebrafish has  proven to be a valuable model system for exploring skeletal muscle function and  for studying human muscle diseases. Despite  the many ...

Intravital Microscopy for Imaging Subcellular Structures in Live Mice Expressing Fluorescent Proteins

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, we use the salivary glands of live mice since they provide several advantages. First, they can be easily exposed to enable access to the optics, and stabilized to facilitate the reduction of the motion artifacts due to heartbeat and respiration. This significantly facilitates imaging and tracking small subcellular structures. Second, most of the cell populations of the salivary glands are accessible from the surface of the organ. This permits the use of confocal microscopy that has a higher spatial resolution than other techniques that have been used for in vivo imaging, such as two-photon microscopy. Finally, salivary glands can be easily manipulated pharmacologically and genetically, thus providing a robust system to investigate biological processes at a molecular level.
In this study we focus on a protocol designed to follow the kinetics of the exocytosis of secretory granules in acinar cells and the dynamics of the apical plasma membrane where the secretory granules fuse upon stimulation of the beta-adrenergic receptors. Specifically, we used a transgenic mouse that co-expresses cytosolic GFP and a membrane-targeted peptide fused with the fluorescent protein tandem-Tomato. However, the procedures that we used to stabilize and image the salivary glands can be extended to other mouse models and coupled to other approaches to label in vivo cellular components, enabling the visualization of various subcellular structures, such as endosomes, lysosomes, mitochondria, and the actin cytoskeleton.

Intravital microscopy is a powerful tool that enables imaging various biological processes in live animals. In this article, we present a detailed method for imaging the dynamics of subcellular structures, such as the secretory granules, in the salivary glands of live mice.

活体显微镜成像的亚细胞结构的活体小鼠表达荧光蛋白

Here we describe a procedure to image subcellular structures in live rodents that is based on the use of confocal intravital microscopy. As a model organ, ...

Live Cell Imaging and 3D Analysis of Angiotensin Receptor Type 1a Trafficking in Transfected Human Embryonic Kidney Cells Using Confocal Microscopy

Live-cell imaging is used to simultaneously capture time-lapse images of angiotensin type 1a receptors (AT1aR) and intracellular compartments in transfected human embryonic kidney-293 (HEK) cells following stimulation with angiotensin II (Ang II). HEK cells are transiently transfected with plasmid DNA containing AT1aR tagged with enhanced green fluorescent protein (EGFP). Lysosomes are identified with a red fluorescent dye. Live-cell images are captured on a laser scanning confocal microscope after Ang II stimulation and analyzed by software in three dimensions (3D, voxels) over time. Live-cell imaging enables investigations into receptor trafficking and avoids confounds associated with fixation, and in particular, the loss or artefactual displacement of EGFP-tagged membrane receptors. Thus, as individual cells are tracked through time, the subcellular localization of receptors can be imaged and measured. Images must be acquired sufficiently rapidly to capture rapid vesicle movement. Yet, at faster imaging speeds, the number of photons collected is reduced. Compromises must also be made in the selection of imaging parameters like voxel size in order to gain imaging speed. Significant applications of live-cell imaging are to study protein trafficking, migration, proliferation, cell cycle, apoptosis, autophagy and protein-protein interaction and dynamics, to name but a few.

Here we present a protocol to image cells expressing green fluorescent protein-tagged angiotensin type 1a receptors during endocytosis initiated by angiotensin II treatment. This technique includes labeling lysosomes with a second fluorescent marker, and then utilizing software to analyze the co-localization of receptor and lysosome in three dimensions over time.