Cold Spring Harb Protoc 2013 Aug 01;20138:768-70. doi: 10.1101/pdb.prot076463.

Dye labeling retinal ganglion cell axons in live Xenopus tadpoles.

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Individual neurons in the developing nervous system can be visualized by the targeted delivery of a fluorophore. In this article, we describe a method for introducing a fluorescent dye via iontophoresis into retinal ganglion cell (RGC) axons in albino Xenopus laevis tadpoles. Iontophoresis is the enhanced permeation of molecules across biological membranes under the influence of an electrical field. Lipophilic dyes such as DiI are well suited to this method--being insoluble in the aqueous environment of the eye, they precipitate instantaneously, and only cells in contact with the dye crystal are labeled as the dye diffuses through the plasma membrane. A dissection stereomicroscope is used to allow a wide range of approach angles for the micropipette. The goal is to introduce a small bolus of dye into the neural retina where the ganglion cell somata are located and the axons course, with the expectation that it will be taken up by a small enough number of axons to allow individual cells to be distinguished. Because RGC axons will typically be imaged in the tectum far from the injection site, a relatively large injection can be made, increasing the probability of labeling axons without obscuring their visualization at the target. This approach is particularly useful under conditions in which it might be too difficult to perform juxtacellular electroporation because of limited visibility or access.

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