Becker Klaus, Jährling Nina, Saghafi Saiedeh, Dodt Hans-Ulrich
Cold Spring Harb Protoc. 2013 Aug 1;2013(8):743-4. doi: 10.1101/pdb.prot076521.
This protocol describes the preparation of mouse embryos for ultramicroscopy (UM), a powerful imaging technique that achieves precise and accurate three-dimensional (3D) reconstructions of intact macroscopic specimens with micrometer resolution. In UM, a specimen in the size range of ∼1-15 mm is illuminated perpendicular to the observation pathway by two thin counterpropagating sheets of laser light. In combination with fluorescein isothiocyanate (FITC) immunostaining, UM allows visualization of somatic motor and sensorial nerve fibers in whole mouse embryos. Even the fine branches of the sensomotoric fibers can be visualized over a distance of up to several millimeters. In this protocol, mouse embryos are fixed and immunostained in preparation for UM. Because UM requires the excitation light sheet to travel throughout the entire horizontal width of the specimen, specimens usually have to be rendered transparent before microscope inspection. Here, the embryos are dehydrated in ethanol and then cleared in a solution of benzyl alcohol and benzyl benzoate.
本方案描述了用于超微显微镜（UM）的小鼠胚胎制备方法，超微显微镜是一种强大的成像技术，能够以微米级分辨率对完整的宏观标本进行精确且准确的三维（3D）重建。在超微显微镜中，大小约为1 - 15毫米的标本由两束反向传播的薄激光片垂直于观察路径进行照明。结合异硫氰酸荧光素（FITC）免疫染色，超微显微镜可使整个小鼠胚胎中的躯体运动和感觉神经纤维可视化。即使是感觉运动纤维的细小分支，在长达数毫米的距离内也能被可视化。在本方案中，小鼠胚胎需进行固定和免疫染色以准备用于超微显微镜观察。由于超微显微镜要求激发光片贯穿标本的整个水平宽度，因此在显微镜检查前通常必须使标本变得透明。在此，胚胎先在乙醇中脱水，然后在苯甲醇和苯甲酸苄酯的溶液中进行透明处理。
