Morgan Josh L, Kerschensteiner Daniel
Cold Spring Harb Protoc. 2011 Dec 1;2011(12):1507-11. doi: 10.1101/pdb.prot067041.
Imaging and reconstruction of developing neurons require cells that are labeled in a way that distinguishes them from their neighbors. This can be achieved with ballistic labeling, which refers to the delivery of a cell label by means of carrier particles (tungsten or gold) propelled from a pressurized gun. Ballistic delivery can reach many dispersed cells in one shot and can deploy a wide variety of cell markers to neurons in diverse preparations. The three most commonly used types of ballistic labels are carbocyanine dyes, dextran-conjugated fluorescent markers, and DNA plasmids. The primary advantage of ballistic labeling is that multiple dispersed cells can be labeled quickly in live or fixed tissue. This article describes a protocol for coating tungsten particles (∼1 µm in diameter) with carbocyanine dyes, which are widely used to label neurons in tissue and neural cells in suspension. These dyes are lipophilic and highly fluorescent within lipid bilayers. Because tissue damage worsens with the increasing pressure required for deeper bullet penetration, ballistic labeling of neurons is most effective when the target cells are near the surface of the preparation. This protocol was developed for labeling ganglion cells in retinal flat mounts.
发育中神经元的成像和重建需要以能将它们与相邻细胞区分开的方式进行标记的细胞。这可以通过弹道标记来实现，弹道标记是指借助从加压枪发射的载体颗粒（钨或金）来传递细胞标记。一次弹道传递就能标记许多分散的细胞，并且可以将多种细胞标记物应用于不同制剂中的神经元。三种最常用的弹道标记类型是碳菁染料、葡聚糖偶联荧光标记物和DNA质粒。弹道标记的主要优点是可以在活组织或固定组织中快速标记多个分散的细胞。本文描述了一种用碳菁染料包被钨颗粒（直径约1 µm）的方法，碳菁染料广泛用于标记组织中的神经元和悬浮液中的神经细胞。这些染料具有亲脂性，在脂质双层中具有高荧光性。由于随着子弹更深穿透所需压力的增加，组织损伤会加剧，因此当靶细胞靠近制剂表面时，神经元的弹道标记最为有效。该方法是为标记视网膜平铺标本中的神经节细胞而开发的。
