Ivins Sarah, Roberts Catherine, Vernay Bertrand, Scambler Peter J
Developmental Biology of Birth Defects, UCL Institute of Child Health;
Developmental Biology of Birth Defects, UCL Institute of Child Health.
J Vis Exp. 2016 Dec 7(118):54800. doi: 10.3791/54800.
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.
由于成像光会被厚实的心脏组织散射，使用标准显微镜技术对胚胎冠状动脉丛（毛细血管和动脉网络将从中形成）进行整体可视化变得困难，因为这些血管位于发育中心脏壁的深处。由于已证明使用诸如BABB（1份苯甲醇与2份苯甲酸苄酯）之类的有机溶剂对组织进行光学透明处理可大大提高可实现的光学穿透深度，因此我们将完整的、经PECAM1免疫染色的心脏的透明处理与激光扫描共聚焦显微镜相结合，以获得整个心脏血管的高分辨率图像。胚胎心脏的BABB透明处理进行得很快，并且还能将荧光信号保存数周；此外，样品可以多次成像而不会丢失信号。这种简单的方法也适用于对整个胚胎中的其他类型血管进行成像。
