这项工作是由美国国家卫生研究院生物医学研究中心在大奥蒙德街儿童医院NHS信托基金会和伦敦大学学院支持英国心脏Foundation&#39;and资助。

所有的动物研究是按照机构准则许可由英国内政部进行。 1.解剖和胚胎红心固定<ol><li>安乐死上使用伦理批准剔除技术， 例如，CO 2麻醉颈椎脱位所需天定时怀孕小鼠，并取出胚胎囊14，将它们放置在一个10cm培养皿充满磷酸盐缓冲盐水（PBS） 。 </li><li>使用解剖显微镜和镊子，小心翼翼地打开了子宫囊，取出每个胚胎，切断脐带和去除卵黄囊。 </li><li>对于基因分型的目的，去除胚胎尾部，地点在裂解/ DNA提取后，一个1.5 ml离心管。 </li><li>为了去除心脏，引脚每个胚胎出其在的PBS填充的硅氧烷弹性体涂覆35毫米培养皿背面，采用不锈钢minutien销。用细镊子，CArefully打开胸腔并切断大血管，前心脏。然后到达身后镊子的心脏，抓住后面的心脏血管和拉轻轻取出心脏;肺部也可能在同一时间一起离开。 </li><li>的心脏转移到含有PBS中新鲜35毫米培养皿，并使用细镊子如果需要修剪掉肺组织。然后将心脏转移到填充有冷PBS一个48孔板的孔中。 </li><li>收集所有的心在48孔板后，吸出PBS用细尖的塑料巴斯德吸管，并用0.5毫升4％多聚甲醛（PFA）取代。 小心:PFA是一种已知的过敏，致癌和有毒的。 <ol><li>修复E11.5 - 12.5心15分钟，E13.5心20分钟，并E14.5 - 15.5心30分钟，在4％PFA室温。 </li></ol></li><li>吸出PFA用细尖的塑料巴斯德吸管和冲洗的心2个冷PBS。 CAUTION:PFA是危险的，并按照规定的制度，必须进行处理。 </li><li>如果不是整个装载免疫立即使用（见第2部分），通过在以下的溶液进行连续5分钟洗涤脱水心中:25％甲醇/ 75％PBS中，50％甲醇/ 50％PBS中，75％甲醇/ 25 PBS％。 注意:甲醇是有毒的，戴手套。在100％甲醇-20℃储存的心。 </li></ol> 2.胚胎心脏的全山免疫染色与反PECAM1抗体注:抗PECAM1抗体工作良好染色的冠状血管（参见步骤2.4），但任何泛内皮标记物，给出了一个可靠的信号是合适的。 <ol><li>如果使用已储存在100％甲醇中心，转移到1.5 ml微量离心管，并通过在下述溶液进行连续5分钟洗涤再水化:75％甲醇/ 25％PBS中，50％甲醇/ 50％PBS中的25％甲醇/ 75％PBS中。 </li&gt; <li> 1.0毫升PBST（PBS / 0.1％吐温20），在室温下旋转 - 通过在0.5执行3×10分钟洗涤通透的心。 </li><li>通过在室温下在在PBST1.0毫升10％山羊血清1小时旋转框的心。 </li><li>用细尖的塑料巴斯德吸管或1000微升枪头，删除封闭液并用400取代 - 含1:50稀释的抗PECAM1一抗500微升新鲜块。于4℃过夜旋转管。 </li><li>第二天，吸块/一抗用细尖的塑料巴斯德吸管或1000微升枪头，并于1.0ml PBST进行至少6倍的1小时清洗，在室温下旋转管。 </li><li>替换用含有稀释1所述荧光团共轭的二级抗体新鲜封闭缓冲液在最后一次洗涤:500，并在4℃下孵育过夜，用旋转。通过在铝箔包装管避光。 </li><li>第二天，吸块/秒用细尖的塑料巴斯德吸管ondary抗体，并进行至少6倍1小时洗于1.0ml PBST，在室温下旋转管。 </li><li>在4℃（避光），直到需要存储的心。 </li></ol> 3.结算解决方案和显微镜菜的制备注意:当在BABB成像心中至关重要的是，没有一个BABB溶液进入与显微镜的部件的接触。为此目的，以下方案包含用于制备适于荧光显微镜硅氧烷弹性体密封的菜肴，可提前做好准备的指示。有机硅弹性体提供一种屏障以包含BABB并防止它从玻璃底部和盘的聚碳酸酯侧面之间潜在的渗漏。 <ol><li>为了准备有机硅弹性体涂层的菜肴，以光学玻璃底培养皿，并将从4毫升螺丝帽小瓶顶部（约1.5公分）在每个培养皿的中心。 注:当弹性体被施加到盘这将形成一个良好的样品。 </li><li>填充的硅氧烷弹性体的菜至大约0.5cm的深度，用涂布器盒，它们被施加到两个组分混合，根据制造商的说明。离开以固化至少24小时，确保瓶盖保持在每个培养皿的中心。 注:使用安全眼镜，以避免眼睛有机硅弹性体的偶然接触。 </li><li>固化该弹性体后，从各皿的瓶盖，这会留下一个中央孔，其中待成像的样品可以被放置在与餐具的玻璃底直接接触。 注:固化后的弹性体应牢固，干燥的触感。 </li><li>制备BABB溶液（1份苄醇2份苯甲酸苄酯）。这应储存在玻璃瓶中并避光。 注意:BABB是有毒的，腐蚀性的溶液。处理在通风橱，而穿相应的防护服。 </li><li>混合BABB和甲醇，制成50:50溶液 - 在玻璃小瓶（1 5ml）中。从光， 如保护，通过包装箔小瓶。 </li></ol> 4.脱水，结算和共聚焦显微镜红心安装注:较大的样品可在4毫升玻璃瓶中被清除，但是对于小样本（ 如，心达E13.5）最好是在显微镜菜直接进行结算的过程。这有助于防止"丢失"的样品一样的心变得很透明，一旦清理。对于这些小样本结算是非常迅速的，在几分钟之内发生。 注:避光样品中通过包装/覆盖管和菜带箔以下所有步骤。 <ol><li>清理前，在室温旋转坦佩心中脱水通过甲醇系列中的免疫染色，心叉涂抹在下列溶液的连续变化的1.5 ml离心管:25％甲醇/ 75％PBS中，50％甲醇/ 50％PBS中和75％甲醇/ 25％PBS中（5分钟在每个）。最后，转动，以便在100％甲醇中3×5分钟。 </li><li>对于心达E13.5，发生在显微镜盘的中心;在显微镜下保证心脏被定向，其背侧浮上心头。来自各地的心脏用细尖的塑料巴斯德吸管取出任何甲醇。 </li><li> （ - 400微升约300个）的足量沉浸在心脏甲醇50:50的解决办法:用干净的细尖塑料巴斯德吸管，添加BABB。由于心中有时可以在溶液空翻多，再次检查方向，而心中仍然不透明。留在5分钟的溶液中。 </li><li>在通风橱中取出50:50解决了玻璃废液瓶和BABB更换，再次使用一个带尖的塑料巴斯德吸管。 注:不需要大容量;添加SUFFicient使心脏位于溶液滴内。该心脏应在5分钟内清除。 </li><li>对于较大的心灵，清除玻璃小瓶样品。 注释:E15.5心脏应该清楚在30分钟，1小时，但如果需要的话可以留过夜。 </li><li>样品是清楚后，取出溶液并用BABB的最小体积替换。任选，盖上盖玻片样品，以帮助保持在适当位置，然而，这不是绝对必要的。使用心脏成像。 </li></ol> 5.成像清除心激光共聚焦显微镜注:图像，使用10X或20X干燥目标倒置共聚焦显微镜拍摄的。尽管有可能使用在一个直立共焦的菜，额外必须小心，以避免与BABB溶液物镜接触。 <ol><li>收集心脏15的Z扫描。根据心脏的大小，瓦片扫描可能需要图像整个心脏16。 <br/&gt; 注意:BABB是一种腐蚀性溶液，戴上干净的手套，并确保显微镜盘的外面是从BABB免费。小心处理的菜，并保持在盘盖，以减少意外泄漏的到显微镜的风险。 </li><li>如果目标工作距离允许，使用较高的放大倍率获得的感兴趣区域的更高分辨率的图像。 </li></ol> 6.数据分析应用软件斐济<ol><li>打开图像的Z堆栈在斐济17。 </li><li>为了使整个堆栈或它的一部分的AZ投影，点击图片并选择堆栈，然后由Z项目。进入启动和停止切片编号和特定的z的投影， 例如，平均强度，最大强度的类型。 </li><li>为了形象片使用吹塑工具的堆栈中彰显个性的船只（从插件菜单中选择分割，然后吹/套索工具），然后单击图像的区域在EA填写CH片。使用填充命令（点击编辑，然后选择填充）与选择的前景色填充所选的区域（点击编辑，然后选择选项，其次是颜色和前景）。 </li><li> ž项目的形象和以前一样叠加。 </li></ol>冠状动脉7.三维曲面体绘制生成使用了Imaris <ol><li>点击超越视图图标在屏幕的顶部和拖动和图像文件拖放到数据窗口。点击三维视图图标在屏幕的顶部。 </li><li>选择面图标（蓝色图标），然后单击创建选项卡上。点击"跳过自动创建，手动编辑"对话框，单击绘图图标（瘦铅笔图标）。 </li><li>选择轮廓选项卡，然后在模式选项卡。在模式中，点击魔棒或等值线图的图标。点击旁边的"选择"选择在屏幕的右侧指针选择指针模式，然后点击"绘制9;框（在屏幕的左下角）。 </li><li>使用等值线或魔棒工具连续切片选择动脉的轮廓。从切片导航使用切片位置滑块片。 </li><li>当所有的轮廓都被选中，单击创建面框。周边体积可以做得不可见，或者使用混合模式呈现或多或少不透明;点击音量选中它，然后选择设置选项卡，其次是模式选项卡。选择Blend和使用滑块以更改透明度。 </li><li>捕获使用快照功能的图像。 </li></ol>

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The authors have nothing to disclose.

在整个胚胎心脏冠状血管通过用抗PECAM1抗体，然后通过光学间隙和共聚焦​​显微镜wholemount免疫染色成像。这里介绍的直接的方法，与BABB胚胎小鼠心脏的间隙，提高了光穿透，使血管中的主动脉和心室壁局部的高清晰度图像的采集。基于甘油安装试剂如的Vectashield（折射率1.45）也已用于冠状脉管22然而BABB的更高的折射率（1.56）进一步减少光散射，使更深的组织穿透的成像。组织间隙避免了更复杂的，昂贵的形式镜如多光子光表镜可能是不太容易提供给研究人员的需要。相比其它方法6-9和用于小样本可以是加利福尼亚州信息交换过程是非常迅速rried了使用直接在显微镜菜试剂的小卷。脉管的鲁棒染色，以实现高质量的图像是必需的;选择抗PECAM1抗体，因为它标志着各类冠状动脉内皮细胞和一些商业抗体被发现给予适当的高水平染色。此外，荧光染色似乎是非常稳定的BABB;在室温下保存在BABB样品（避光）保持其荧光信号数月。该PECAM1抗体有效地标记在冠状心内膜以及脉管是偶尔有问题的，捕捉peritruncal丛图像特别是当这一事实。相比于peritruncal内皮主动脉管腔的强染色导致过饱和的图像中的一些区域的危险，这意味着被要求的摄像参数仔细调整。理想地，抗体染色仅血管内皮细胞将被使用;在实践中，然而，发现产率wholemount染色所要求的水平适当的抗体是困难的。最近，脂肪酸结合蛋白4（FABP4）已被证明是冠状血管内皮23的标记物，因此可代表至PECAM1的替代方法。 为了保留的主动脉和心脏腔室中的样品不是平面地安装在三维形态，但在玻璃底培养皿中，而不是成像。样品的待成像的深度排除了使用高倍率物镜，由于它们的短的工作距离。然而高分辨率图像仍可以实现使用10X物镜通过增加像素停留时间，并使用中的至少1024x1024个像素阵列尺寸为图像捕获。这是足够的结构和冠状血管的分布的分析，然而蜂窝结构的更精细的分析，可能需要样品的平面式安装。安装心脏的各个部分的解剖，例如，心室壁，或主动脉，也可能是必要的。备选地，过采样的图像随后去卷积的可能，以增加分辨率和灵敏度进行;然而，这需要相当长的扫描时间，创建需要大量的计算能力来处理的非常大的图像文件。 心至E15.5使用这种方法被成功地成像，并且也可以使用相同的协议来分析整个胚胎的脉管（高达至少E11.5）。其它类型的细胞， 例如，平滑肌细胞也已在我们的实验室使用这种技术成像。对于较厚的组织， 例如，心比E15.5老，抗体渗透可以是一个限制因素;较长的孵育和/或增加的洗涤剂，可能需要。此外，采集图像的大Z堆栈时信号作为激光穿透组织的最远的部分强度降低;然而，通过confOCAL设定可以调节，以增加激光功率与z增加距离。 这种方法有利于两个冠状血管形成的早期阶段，并在以后的发展阶段的冠状动脉的图案形成的共焦显微镜分析。有关的分布，支化和血管的结构的详细信息可以在很短的时间内获得的，使这种用于与血管发生途径的特定缺陷的基因小鼠突变体的研究的有价值的工具。

建立一个有效的冠状动脉网络是心脏功能和胚胎发育和遗传小鼠突变的分析是至关重要的可以提供有价值的洞察潜在的这一发展过程中的分子信号。可视化的胚胎冠状动脉丛作为一个整体的能力，而不是在一系列组织切片的呈现，是必不可少的，以促进在基因突变体容器图案的分析，并且避免信息的潜在损失可能发生由于机械的结果的组织的切片。注定要形成的动脉血管和毛细血管的心室和主动脉1-3两者的城墙内深本地化。然而，虽然细胞用激光扫描共聚焦显微镜相结合，可提供wholemount标记浅静脉/淋巴管4,5的高清晰度的图像的荧光标记，成像的深度由光学穿透限制。帽的高分辨率成像因此，整个心脏的整个深度illaries和动脉是不可能没有某种形式的组织清算。 差光学穿透由多种细胞和细胞外（ 如，胶原和弹性纤维）厚组织的成分的高折射率所致。这种散射成像光，造成模糊和对比度下降。清除剂通常会与这些组织的高折射率，这意味着光可以通过样品通畅旅行和更深地穿透该组织。结算之前，组织通常是脱水水具有相对低的折射率。的新的清算方法的过多最近已开发的，但是这取决于所使用的，结算过程可能需要数天或数周，并且可能需要昂贵的试剂6-9的技术。 BABB（1:苯甲醇和苯甲酸苄酯的1:2混合物）是一种廉价的，常用的清除剂，其具有优势非常快清除样本。基于BABB结算和成像技术已用于神经样品和各种器官10-13如前所述。在这里，我们描述了样品的免疫染色后共聚焦显微镜的清除BABB一个强大的和简单的技术，具体参照血管鼠心从E（胚胎天）11.5检验 - 15.5。然而，如也已经证实，该技术可以同样很好地适用于全胚胎的分析，以及其他类型的细胞，只要是高品质的抗体对感兴趣的标志物是可用的。

<table><tbody><tr><td>PBS</td><td>Life Technologies</td><td>14190-094</td><td></td></tr><tr><td>Forceps</td><td>FST</td><td>11251-30</td><td></td></tr><tr><td>10 cm Petri dishes</td><td>Falcon</td><td>351029</td><td></td></tr><tr><td>35 mm Petri dishes</td><td>Sigma</td><td>P5112</td><td></td></tr><tr><td>Stainless steel minutien pins 0.2 mm diameter</td><td>FST</td><td>26002-20</td><td></td></tr><tr><td>Fine tip pastettes&amp;nbsp;</td><td>Alpha Laboratories</td><td>LW4061</td><td></td></tr><tr><td>1,000 ml pipette tips</td><td>Sorenson</td><td>34000</td><td></td></tr><tr><td>48-well plate</td><td>Falcon</td><td>353078</td><td></td></tr><tr><td>Kwik-Gard</td><td>World Precision Instrument</td><td>KWIKGARD</td><td>Silicone elastomer Sylgard184 packaged in a cartridge for mixing and dispensing</td></tr><tr><td>Kwik-Gard refill</td><td>World Precision Instrument</td><td>KWIKGLUE</td><td>Refill cartridges and dispensing tips</td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td>Make up in PBS and store at -20 &amp;deg;C</td></tr><tr><td>100% methanol</td><td>VWR</td><td>20847307</td><td></td></tr><tr><td>Tween&amp;reg;20</td><td>Sigma</td><td>P1379</td><td></td></tr><tr><td>Goat serum</td><td>Sigma</td><td>G9023</td><td></td></tr><tr><td>Anti-PECAM1 antibody, rat anti-mouse</td><td>BD Pharmingen</td><td>553370</td><td>Primary antibody, dilute 1 in 50</td></tr><tr><td>Anti-CD31 polyclonal, rabbit polyclonal</td><td>Abcam</td><td>ab28364</td><td>Primary antibody, dilute 1 in 50</td></tr><tr><td>Anti-CD31/PECAM1 clone 2H8, armenian hamster monoclonal</td><td>Thermo Fisher Scientific</td><td>MA3105</td><td>Primary antibody, dilute 1 in 400</td></tr><tr><td>Endomucin antibody (V.7C7), rat monoclonal</td><td>Santa Cruz Biotechnology</td><td>sc-65495</td><td>Primary antibody, dilute 1 in 50</td></tr><tr><td>Anti-SM22 alpha antibody, rabbit polyclonal</td><td>Abcam</td><td>ab14106</td><td>Primary antibody, dilute 1 in 250</td></tr><tr><td>Goat anti-rat IgG (H+L)Alexa Fluor 594</td><td>Thermo Fisher Scientific</td><td>A11007</td><td>Secondary antibody, dilute 1 in 500</td></tr><tr><td>Goat anti-rabbit IgG (H+L)Alexa Fluor 594</td><td>Thermo Fisher Scientific</td><td></td><td>Secondary antibody, dilute 1 in 500</td></tr><tr><td>Goat anti-Armenian Hamster IgG (H+L)Alexa Fluor 488</td><td>Abcam</td><td>ab173003</td><td></td></tr><tr><td>Goat anti-rat IgG (H+L)Alexa Fluor 488</td><td>Thermo Fisher Scientific</td><td>A21208</td><td>Secondary antibody, dilute 1 in 500</td></tr><tr><td>Phenolic screw cap</td><td>Wheaton</td><td>240408</td><td></td></tr><tr><td>Benzyl alcohol</td><td>Sigma</td><td>402834</td><td></td></tr><tr><td>Benzyl benzoate</td><td>Sigma</td><td>B6630</td><td></td></tr><tr><td>Imaris</td><td>Bitplane Imaging</td><td>image analysis software</td><td></td></tr><tr><td>ImageJ software</td><td>NIH</td><td>Freeware</td><td></td></tr></tbody></table>

analysis of coronary vessels in cleared embryonic hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

当心脏的发展，心肌由来自各种来源的内皮细胞（EC）侵入，并且形成一个血管丛。心室心肌内制定船只将继续形成毛细血管和动脉网络上提供的含氧血液到心脏组织18。在围绕主动脉14,19,20的内腔的同时（约E11.5）冠状动脉梗开始从单独的毛细管网络（称为peritruncal丛）独立地发展。 Peritruncal丛内皮最初形成在阀窦的水平主动脉管腔多个连接，但最终只有一个容器将持续存在于主动脉的每一侧，将上，形成左和右冠状动脉21的根部。大约从E13.5的peritruncal和室心肌血管联合起来，形成一个相互联系的网络，允许从血流奥尔塔进入冠状血管14,22。抗PECAM-1抗体，与完整的心BABB间隙结合内皮细胞染色，允许开发冠状网络从最早阶段的分析。在后来的发育阶段也可以检查了成熟冠状动脉的图案化。也可以利用这种方法来染色全胚胎的脉管（最多E11.5至少），并用不同的抗体， 例如，抗α-平滑肌肌动蛋白（SM22α）和Endomucin。 图1显示了使用斐济软件生成的E11.5心脏的最大强度Z-预测。平铺功能来收集心脏的多个部分图像和它们拼接成一幅完整的图像;这是非常有用的，得到染色的概述在整个样品。在这个阶段PECAM1抗体染色为主的心脏流出诉心内膜衬essels，然而几内皮，也可以在左室壁观察（盒装区域在图 1A中，示出放大在1A&#39;）。此外，peritruncal丛可以清楚地在流出道（OT）（在图1B盒装区域）的壁观察。在后一种情况下，减少了用于产生投影的Z堆叠片的数目提高了图像的清晰度，允许与主动脉管腔的连接被更清楚地显现（ 图1B&#39;）。在图2中，增加的脉管近端主动脉可以在E12.5心脏中观察到。 图3示出了在E12.5心脏的peritruncal丛。在图3A的12片Ž投影示出了整个丛，但是因为一些血管腹侧定位于主动脉（也很大程度上受到抗PECAM1抗体染色）的内腔，分裂的Z堆叠我n要多个子栈（ 图3B-D）提供了更多的可视信息。例如，所述亚电池堆在图3B投影示出在连接到主动脉管腔的腹面，这不是在全投影可见peritruncal容器中。 图4示出了E15.5心脏的一部分;冠状动脉在此阶段（ 图4A，30切片投影）清晰可见。在图4B中所示的5片Ž突起和C主动脉和肺动脉瓣也可以通过PECAM1染色进行可视化;树枝和冠状动脉毛细血管网的互连，在这些更小的子栈预测还清晰。手工分割技术已被用来突出显示使用斐济（ 图4D）或了Imaris（ 图4D和E）冠状动脉。冠状动脉三维表面体绘制/主动脉腔产生ū唱了Imaris可以使用或不使用周围脉管系统（ 图4E和F）进行查看。 全胚胎的脉管也可以使用PECAM1染色/ BABB间隙检测。分别的胚胎和左侧（Z片66-110） - 图 4A和A&#39;，示出了从右侧从亚电池堆产生的Z-突起（65Ž片11）。两个图像的比较表明，荧光信号的强度没有显著与更深地穿透到组织中降低。在图4B中 ，PECAM1（红色信号）和Endomucin（绿色信号）抗体结合分别以标记一个E11.5胚胎的intersomitic动脉（国际检索单位）和静脉。 图4C示出SM22α（红色信号），并在E11.5胚胎的ISA的PECAM1染色（绿色信号）。在图6中 ，E11.5胚胎与PECAM 1染色被BABB间隙（ 图 4A）之后立即成像，或约2个月（ 图4B）的时间间隔后。荧光信号仍然足够强的图像后成功BABB样品长时间存放。 <img alt="图1" src="/files/ftp_upload/54800/54800fig1.jpg" /> 图1.免疫染色的E11.5心与反PECAM1抗体（与抗大鼠IgG共轭检测到Alexa的594）。 （A，B）2×2的瓷砖图像使用倒置共焦显微镜的10X物镜收集。 PECAM1抗体染色都心内膜和血管内皮细胞。突起（最大强度）从图22（A）或图5（B）z各自4微米厚的切片生成，使用斐济软件。 A&#39;和B&#39;是enlargemen分别在A和B的盒装地区的TS。在A&#39;箭头表示血管中的室壁;在B&#39;，托架表示peritruncal丛。 OT，流出道; LV，左心室，右心室右心室。所有的图片都是正面的看法。在扩展酒吧和B = 200微米;比例尺在A&#39;和B&#39;= 100微米。板1B"改编自艾文斯等人 ，2015年19。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54800/54800fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/54800/54800fig2.jpg" /> 图2.免疫染色与反PECAM1抗体的E12.5心。图A显示了一个2x2拼接扫描AZ投影（最大强度）。 A中的盒装区域显示enlargED在A&#39;;箭头指示多个血管近端主动脉（AO）。 LV，左心室，右心室右心室。所有的图片都是正面的看法。在比例尺= 200微米;比例尺在A&#39;= 100微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54800/54800fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54800/54800fig3.jpg" /> 图3. Peritruncal丛成像在E12.5心。使用10倍的目标收集与抗PECAM1抗体染色的E12.5主动脉（AO）的共聚焦图像。在一个 Z轴投影（最大强度）由12片ž（4微米厚）产生的;箭头指示peritruncal丛血管。 BD 12ż切片分成三个亚电池堆所指示，并用于产生单独projecti附件;箭头指示由peritruncal丛形成到主动脉的内腔连接。腹侧定位于主动脉的内腔Peritruncal容器是在B和C中可见。所有的图片都是正面的看法。比例尺= 50微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54800/54800fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/54800/54800fig4.jpg" /> 图4.冠状动脉及毛细血管丛在E15.5心。使用10倍的目标收集的E15.5心脏抗PECAM1抗体染色的共聚焦图像。图A显示了从30ž切片（4微米厚）产生的最大强度投影ž;箭头表示左和右冠​​状动脉（LCA和RCA）由此可以看出连接到主动脉（AO）。使用differen吨5ž切片亚电池堆的主动脉和肺动脉瓣（AOV和PV）可以在B和C分别（蓝色括号中）可以看到。心室毛细血管丛在这些更小的子栈预测还更清晰;血管近端肺动脉瓣（小盒）的放大显示面板C（大盒）的右下角。斐济是用来彰显个性的血管， 例如 ，用于面板D中的吹瓶/套索工具使用手动用红色填充冠状动脉投影之前，每个Z片。软件了Imaris用于创建动脉（红色）和E和F主动脉腔（绿色）的3D图像;再次这是手工完成，使用魔术棒工具创建的每个Z切片对象的轮廓。周围体积的不透明度可在混合模式被改变，如在电子商务 。可替换地，3D图像可以提取，如图F中</s仲&gt;。 LV，左心室。所有的图片都是正面的看法。比例尺在A = 100微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54800/54800fig4large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图5" src="/files/ftp_upload/54800/54800fig5.jpg" /> 图5.成像胚胎血管。图A和抗PECAM1抗体染色的E10.5的野生型胚，用10倍的目标（平铺扫描）收集到的"节目共聚焦图像。从120ž切片扫描（4微米厚片）中的Z片11-65（A）和66-110（A&#39;），呈现出跨胚厚度荧光强度只有轻微的损失生成平均强度ž预测。 B组显示的E11.5胚胎与antibod染色intersomitic船只<html>针对PECAM1（从594缀合的二级抗体红色信号）和Endomucin（厂房专供从488缀合的二抗绿色信号），IES分别染色intersomitic动脉（箭头）和静脉（箭头）（平均强度Ž投影从平铺扫描）。面板C显示了从与抗体染色针对PECAM1一个E11.5的野生型胚胎intersomitic动脉（国际检索单位）（从488缀合的二抗绿色信号）和SM22α（从594缀合的二级抗体红色信号）。使用20X干物镜收集并用于产生最大强度ž突起共焦图象。所有图像都矢状意见。在A和B比例尺= 250微米，在C =100μm的比例尺。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54800/54800fig5large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> D / 54800 / 54800fig6.jpg"/&gt; 图6.在BABB清零免疫染色样品保留荧光信号为至少两个月。 E11.5胚胎用PECAM1抗体染色，并用BABB清零;从同批胚胎立即或两个月的时间间隔后成像。面板A显示了胚胎的intersomitic动脉立即成像和面板B显示了胚胎成像两个月后。使用10X干物镜收集并用于产生最大强度ž突起共焦图象。道，背主动脉。图片显示矢状意见。在扩展酒吧和B = 100微米。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54800/54800fig6large.jpg" target="_blank">请点击此处查看该图的放大版本。</a>

Watch this Scientific Journal Video about Analysis of Coronary Vessels in Cleared Embryonic Hearts at JoVE.com

Analysis of Coronary Vessels in Cleared Embryonic Hearts

我们提出了一个协议，用于冠状血管的全胚胎小鼠心脏高达E15.5分析，使用标准的免疫染色方法，其次是光学间隙和共聚焦​​显微镜。这种技术使整个心脏血管的可视化，而无需连续切片的耗时的分析。

在清除胚胎心脏冠状血管分析

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

analysis-of-coronary-vessels-in-cleared-embryonic-hearts

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

8.1K 次观看.  UCL Institute of Child Health. 我们提出了一个协议，用于冠状血管的全胚胎小鼠心脏高达E15.5分析，使用标准的免疫染色方法，其次是光学间隙和共聚焦​​显微镜。这种技术使整个心脏血管的可视化，而无需连续切片的耗时的分析。 

视频: Analysis of Coronary Vessels in Cleared Embryonic Hearts

A Method for Labeling Vasculature in Embryonic Mice

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in the thymus proper vasculature formation and patterning is essential for thymocyte entry into the organ and mature T-cell exit to the periphery. The spatial arrangement of blood vessels in the thymus is dependent upon signals from the local microenvironment, namely thymic epithelial cells (TEC). Several recent reports suggest that disruption of these signals results in thymus blood vessel defects 1,2. Previous studies have described techniques used to label the neonatal and adult thymus vasculature 1,2. We demonstrate here a technique for labeling blood vessels in the embryonic thymus. This method combines the use of FITC-dextran or Griffonia (Bandeiraea) Simplicifolia Lectin I (GSL 1 - isolectin B4) facial vein injections and CD31 antibody staining to identify thymus vascular structures and PDGFR-&beta; to label thymic perivascular mesenchyme 3-5. The option of using cryosections or vibratome sections is also provided. This protocol can be used to identify thymus vascular defects, which is critical for defining the roles of TEC-derived molecules in thymus blood vessel formation. As the method labels the entire vasculature, it can also be used to analyze the vascular networks in multiple organs and tissues throughout the embryo including skin and heart 6-10.

This article describes a method for labeling embryonic skin and thymus blood vessels.

标签胚胎小鼠血管的方法

The establishment of a functional blood vessel network is an essential part of organogenesis, and is required for optimal organ function. For example, in ...

科研

免疫与感染

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

成像清除胚胎和出生后心脏在单细胞分辨率

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

发育生物学

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

胚胎血管生成的可视化和定量分析<em&gt;热带非洲爪蟾</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic lamellae and smooth muscle cells (SMCs), and an outer layer of fibroblasts and extracellular matrix. In contrast to the widespread study of pathological models (e.g., atherosclerosis) in the adult aorta, much less is known about the embryonic and perinatal aorta. Here, we focus on SMCs and provide protocols for the analysis of the morphogenesis and pathogenesis of embryonic and perinatal aortic SMCs in normal development and disease. Specifically, the four protocols included are: i) in vivo embryonic fate mapping and clonal analysis; ii) explant embryonic aorta culture; iii) SMC isolation from the perinatal aorta; and iv) subcutaneous osmotic mini-pump placement in pregnant (or non-pregnant) mice. Thus, these approaches facilitate the investigation of the origin(s), fate, and clonal architecture of SMCs in the aorta in vivo. They allow for modulating embryonic aorta morphogenesis in utero by continuous exposure to pharmacological agents. In addition, isolated aortic tissue explants or aortic SMCs can be used to gain insights into the role of specific gene targets during fundamental processes such as muscularization, proliferation, and migration. These hypothesis-generating experiments on isolated SMCs and the explanted aorta can then be assessed in the in vivo context through pharmacological and genetic approaches.

Using In Vivo and Tissue and Cell Explant Approaches to Study the Morphogenesis and Pathogenesis of the Embryonic and Perinatal Aorta

Protocols for studying the embryonic and perinatal murine aorta using in vivo clonal analysis and fate mapping, aortic explants, and isolated smooth muscle cells are detailed here. These diverse approaches facilitate the investigation of the morphogenesis of the embryonic and perinatal aorta in normal development and the pathogenesis in disease.

使用体内和组织和细胞外植体方法研究胚胎和围产期主动脉的形态和发病机制

The aorta is the largest artery in the body. The aortic wall is composed of an inner layer of endothelial cells, a middle layer of alternating elastic ...

Quantitative Whole-mount Immunofluorescence Analysis of Cardiac Progenitor Populations in Mouse Embryos

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development and organogenesis are two areas of research that have benefitted greatly from these advances, due to the high quality of data that can be obtained directly from imaging pre-implantation embryos or ex vivo organs. While pre-implantation embryos have yielded data with especially high spatial resolution, later stages have been less amenable to three-dimensional reconstruction. Obtaining high-quality 3D or volumetric data for known embryonic structures in combination with fate mapping or genetic lineage tracing will allow for a more comprehensive analysis of the morphogenetic events taking place during embryogenesis.
This protocol describes a whole-mount immunofluorescence approach that allows for the labeling, visualization, and quantification of progenitor cell populations within the developing cardiac crescent, a key structure formed during heart development. The approach is designed in such a way that both cell- and tissue-level information can be obtained. Using confocal microscopy and image processing, this protocol allows for three-dimensional spatial reconstruction of the cardiac crescent, thereby providing the ability to analyze the localization and organization of specific progenitor populations during this critical phase of heart development. Importantly, the use of reference antibodies allows for successive masking of the cardiac crescent and subsequent quantitative measurements of areas within the crescent. This protocol will not only enable a detailed examination of early heart development, but with adaptations should be applicable to most organ systems in the gastrula to early somite stage mouse embryo.

Here, we describe a protocol for whole mount immunofluorescence and image-based quantitative volumetric analysis of early stage mouse embryos. We present this technique as a powerful approach to qualitatively and quantitatively assess cardiac structures during development, and propose that it may be widely adaptable to other organ systems.

小鼠胚胎心肌内祖群的定量全贴免疫荧光分析

The use of ever-advancing imaging techniques has contributed broadly to our increased understanding of embryonic development. Pre-implantation development ...

Analysis of Cardiac Chamber Development During Mouse Embryogenesis Using Whole Mount Epifluorescence

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during heart development using ventricular specific fluorescent reporter knock-in mice (MLC-2v-tdTomato mice). Heart development involves a linear heart tube formation, the heart tube looping, and four chamber septation. These complex processes are highly conserved in all vertebrates. The mouse embryonic heart has been widely used for heart developmental studies. However, due to their extremely small size, dissecting mouse embryonic hearts is technically challenging. In addition, visualization of cardiac chamber formation often needs in situ hybridization, beta-galactosidase staining using LacZ reporter mice, or immunostaining of sectioned embryonic hearts. Here, we describe how to dissect mouse embryonic hearts and directly visualize ventricular chamber formation of MLC-2v-tdTomato mice using whole mount epifluorescent microscopy. With this method, it is possible to directly examine heart tube formation and looping, and four chamber formation without further experimental manipulation of mouse embryos. Although the MLC-2v-tdTomato reporter knock-in mouse line is used in this protocol as an example, this protocol can be applied to other heart-specific fluorescent reporter transgenic mouse lines.

We present the protocols to examine mouse heart development using whole mount epifluorescent microscopy on mouse embryos dissected from ventricular specific MLC-2v-tdTomato reporter knock-in mice. This method allows us to directly visualize each stage of the ventricular formation during mouse heart development without labor-intensive histochemical methods.

全山荧光在小鼠胚胎发生过程中心脏室发育的分析

The goal of this protocol is to describe a method for the dissection of mouse embryos and visualization of embryonic mouse ventricular chambers during ...

Analysis of Congenital Heart Defects in Mouse Embryos Using Qualitative and Quantitative Histological Methods

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes for CHD are still poorly understood. The developing mouse constitutes a valuable model for the study of CHD, because cardiac developmental programs between mice and humans are highly conserved. The protocol describes in detail how to produce mouse embryos of the desired gestational stage, methods to isolate and preserve the heart for downstream processing, quantitative methods to identify common types of CHD by histology (e.g., ventricular septal defects, atrial septal defects, patent ductus arteriosus), and quantitative histomorphometry methods to measure common muscular compaction phenotypes. These methods articulate all the steps involved in sample preparation, collection, and analysis, allowing scientists to correctly and reproducibly measure CHD.

In this protocol, we describe procedures to qualitatively and quantitatively analyze developmental phenotypes in mice associated with congenital heart defects.

使用定性和定量组织方法对小鼠胚胎先天性心脏缺陷的分析

Congenital heart defects (CHD) are the most common type of birth defect in humans, affecting up to 1% of all live births. However, the underlying causes ...

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart disease. The development of the primitive cardiac valves involves the epithelial-to-mesenchymal transition (EMT) of endocardial cells from the atrioventricular canal (AVC) and outflow tract (OFT) regions of the heart in response to local inductive myocardial and endocardial signals. Once the cells delaminate and invade the extracellular matrix (cardiac jelly) located between the endocardium and the myocardium, the primitive endocardial cushions (EC) are formed. This process implies that the endocardium has to fill the gaps left by the delaminated cells and has to reorganize itself to converge (narrow) or extend (lengthen) along an axis. Current research has implicated the planar cell polarity (PCP) pathway in regulating the subcellular localization of the factors involved in this process. Classically, the initial phases of cardiac valve development have been studied in cross-sections of embryonic hearts or in ex vivo AVC or OFT explants cultured on collagen gels. These approaches allow the analysis of apico-basal polarity but do not allow the analysis of cell behavior within the plane of the epithelium or of the morphological changes of migrating cells. Here, we show an experimental approach that allows the visualization of the endocardium at valvulogenic regions as a planar field of cells. This experimental approach provides the opportunity to study PCP, planar topology, and intercellular communication within the endocardium of the OFT and AVC during valve development. Deciphering new cellular mechanisms involved in cardiac valve morphogenesis may contribute to understanding congenital heart disease associated with endocardial cushion defects.

En Face Endocardial Cushion Preparation for Planar Morphogenesis Analysis in Mouse Embryos

Classically, the endocardium of the mouse embryonic valve primordium has been analyzed using transversal, coronal, or sagittal sections. Our novel approach for en face, two-dimensional imaging of the endocardium at valvulogenic regions allows planar polarity and cell rearrangement analysis of the endocardium during valve development.

En Face 用于小鼠胚胎平面形态发生分析的心内膜垫制备

The study of the cellular and molecular mechanisms underlying the development of the mammalian heart is essential to address human congenital heart ...

A Pipeline to Characterize Structural Heart Defects in the Fetal Mouse

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe CHD died before adulthood, with the maximum mortality during the first week of life. Remarkable advances in surgical techniques, diagnostic approaches, and medical management have led to marked improvements in outcomes. To address the critical research needs of understanding congenital heart defects, murine models have provided an ideal research platform, as they have very similar heart anatomy to humans and short gestation rates. The combination of genetic engineering with high-throughput phenotyping tools has allowed for the replication and diagnosis of structural heart defects to further elucidate the molecular pathways behind CHDs. The use of noninvasive fetal echocardiography to screen the cardiac phenotypes in mouse models coupled with the high fidelity of Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) histopathology with three-dimensional (3D) reconstructions enables a detailed view into the anatomy of various congenital heart defects. This protocol outlines a complete workflow of these methods to obtain an accurate diagnosis of murine congenital heart defects. Applying this phenotyping protocol to model organisms will allow for accurate CHD diagnosis, yielding insights into the mechanisms of CHD. Identifying the underlying mechanisms of CHD provide opportunities for potential therapies and interventions.

This article details murine congenital heart disease (CHD) diagnostic methods using fetal echocardiography, necropsy, and Episcopic fluorescence image capture (EFIC) using Episcopic confocal microscopy (ECM) followed by three-dimensional (3D) reconstruction.

表征胎儿小鼠结构性心脏缺陷的管道

Congenital heart diseases (CHDs) are major causes of infant death in the United States. In the 1980s and earlier, most patients with moderate or severe ...

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. Thus, protocols to isolate and culture individual organs of interest are essential to provide a method of both visualizing changes in development and allowing novel treatment strategies. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel. This substrate provides enough support to maintain the three-dimensional structure but is flexible enough to allow continued contraction. In brief, hearts are excised from the embryo and placed in a mixture of cold Matrigel diluted 1:1 with growth medium. After the diluted Matrigel solidifies, growth medium is added to the culture dish. Hearts excised as late as embryonic day 16.5 were viable for four days post-dissection. Analysis of the coronary plexus shows that this method does not disrupt coronary vascular development. Thus, we present a novel method for long-term culture of embryonic hearts.

A Novel Ex vivo Culture Method for the Embryonic Mouse Heart

Developmental studies in the mouse are hampered by the inaccessibility of the embryo during gestation. To promote the long-term culture of the embryonic heart at late stages of gestation, we developed a protocol in which the excised heart is cultured in a semi-solid, dilute Matrigel.

一种新型的<em&gt;体外</em小鼠胚胎心脏的培养方法

Developmental studies in the mouse are hampered  by the inaccessibility of the embryo during gestation. Thus, protocols to  isolate and culture individual ...