Pu M, Berson D M
Division of Biology and Medicine, Brown University, Providence, RI 02912.
J Neurosci Methods. 1992 Jan;41(1):45-51. doi: 10.1016/0165-0270(92)90122-t.
We have developed a method for reliable, permanent, high-resolution intracellular staining of ganglion cells in mammalian retinas. Living ganglion cells in the isolated retina are impaled in vitro and injected intracellularly with both Lucifer Yellow (LY) and biocytin. After fixation and aggressive pretreatment of the retina with detergents, the LY is tagged immunohistochemically with biotin using a commercially available anti-LY antibody and a biotinylated secondary antibody. A conventional avidin-biotin procedure is then used to visualize both the biocytin and the biotinylated bridge antibody, yielding complete Golgi-like filling of the soma, dendrites and axon. Advantages of the method include the ease and speed of dye injection, the reliable recovery of stained cells, the large number of cells which can be stained in single retinas, and the high resolution and permanence of the stain, which permit prolonged examination and quantitative analysis.
我们已经开发出一种用于对哺乳动物视网膜神经节细胞进行可靠、持久、高分辨率细胞内染色的方法。将分离视网膜中的活神经节细胞在体外进行刺入，并向细胞内注射荧光黄（LY）和生物胞素。在固定视网膜并用去污剂进行强力预处理后，使用市售的抗LY抗体和生物素化二抗通过免疫组织化学方法用生物素标记LY。然后采用传统的抗生物素蛋白-生物素程序来可视化生物胞素和生物素化的桥抗体，从而实现对胞体、树突和轴突的完全高尔基样填充。该方法的优点包括染料注射简便快捷、染色细胞回收可靠、单个视网膜中可染色的细胞数量多，以及染色的高分辨率和持久性，这使得能够进行长时间检查和定量分析。
