Clanton Joshua A, Shestopalov Ilya, Chen James K, Gamse Joshua T
Department of Biological Sciences, Vanderbilt University, USA.
J Vis Exp. 2011 Apr 28(50):2672. doi: 10.3791/2672.
A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling and pressure injection of traceable enzymes to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularity attractive tools for lineage labeling and to observe the migration of cells in vivo. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequently, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.
发育生物学中的一个核心问题是推断脊椎动物中众多细胞类型从未分化前体产生时的起源。研究人员采用了各种谱系标记方法，如DiI标记和可追踪酶的压力注射，以确定模型系统发育后期的细胞命运。斑马鱼（Danio rerio）中的第一张命运图谱是通过将荧光染料（如罗丹明葡聚糖）离子电渗注入胚胎离散区域的单个细胞，并随时间追踪标记细胞的命运而绘制的。虽然这些方法有效，但技术要求高，需要斑马鱼实验室中不常见的专门设备。最近，光转换荧光蛋白，如Eos和Kaede，在暴露于紫外光时会不可逆地从绿色荧光转换为红色荧光，在斑马鱼中的使用越来越多。斑马鱼胚胎的光学清晰度和相对容易的转基因操作使这些特殊的工具成为谱系标记和观察体内细胞迁移的有吸引力的工具。尽管它们有用，但与染料介导的谱系标记方法相比，这些蛋白有一些缺点。最关键的是我们发现在这些蛋白的光转换过程中难以获得高三维分辨率。有鉴于此，也许在斑马鱼中进行谱系标记时分辨率和易用性的最佳组合是使用笼锁荧光素葡聚糖，这是一种荧光染料，与一个淬灭基团结合，该淬灭基团会掩盖其荧光。然后可以使用来自激光或汞灯的紫外光在特定细胞内将染料“解锁”（从淬灭基团释放），从而使其荧光可视化或进行免疫检测。与离子电渗方法不同，笼锁荧光素可以用标准注射设备注射，并用配备针孔的落射荧光显微镜解锁。此外，针对荧光素的抗体仅检测解锁形式，并且表位在固定后保存良好。最后，笼锁荧光素可以以非常高的三维分辨率激活，特别是如果采用双光子显微镜。本方案描述了一种通过笼锁荧光素和激光解锁进行谱系标记的方法。随后，通过抗体标记，将解锁的荧光素与其他表位（如GFP）同时检测。
