A method for reliable and permanent intracellular staining of retinal ganglion cells

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.