A method for reliable and permanent intracellular staining of retinal ganglion cells
A method for reliable and permanent intracellular staining of retinal ganglion cells
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DOI：
10.1016/0165-0270(92)90122-t
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发表时间：
1992-01
影响因子：
3
通讯作者：
M. Pu;D. Berson
中科院分区：
文献类型：
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作者：
M. Pu;D. Berson
我们开发了一种对哺乳动物视网膜神经节细胞进行可靠、永久、高分辨率细胞内染色的方法。在体外刺穿离体视网膜中的活神经节细胞，并在细胞内注射荧光黄 (LY) 和生物胞素。用去污剂对视网膜进行固定和积极预处理后，使用市售的抗 LY 抗体和生物素化二抗对 LY 进行生物素免疫组织化学标记。然后使用传统的亲和素-生物素程序来可视化生物胞素和生物素化桥抗体，从而产生体体、树突和轴突的完整高尔基样填充。该方法的优点包括染料注射方便快捷、染色细胞的可靠回收、单个视网膜上可染色的大量细胞以及染色的高分辨率和持久性，从而允许长时间检查和定量分析。
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.