Journal of Neuroscience Methods, 32 (1990) 235-243 235 Elsevier NSM 01077 A 'puff and advance' technique for visually controlled staining of turtle retinal ganglion cells Josef AmmermUller 1, Gloria Guiloff 2, Richard Normann 2.3 and Helga Kolb 2 I Dept. of Neurobiology, University of Oldenburg, Oldenburg (F.R.G.), 2 Department of Physiology', School of Medicine, University of Utah, and s Department of Bioengineering, Universi(v of Utah, Salt Lake CiO,, UT 84108 (U.S.A.) (Received 9 November 1989) (Revised version received 9 February 1990) (Accepted 10 February 1990) Key words: Retina; Ganglion cells; Staining; Lucifer yellow; Rhodamine; Horseradish peroxidase; Turtle We describe a 'puff and advance' technique for visually controlled staining of retinal ganglion cells (GCs) in the unfixed, living retina for light and electron microscopy. Glass microelectrodes are filled with rhodamine-isothiocyanate labeled horseradish peroxidase (Rh-HRP), or Lucifer yellow (LY), or a mixture of both, or with 5,6-carboxytetramethylrhodamine (5,6-Rh) and advanced tangentially through the GC layer with microscopic observation using epifluorescence. Brief "puffs" of LY or 5,6-Rh are constantly ejected from the advancing electrode tip by a train of negative current pulses. GC penetration is signaled by virtually instantaneous staining of its soma (and eventually its axon and dendrites if the electrode is not advanced further). An impaled GC can be electron densely stained with the Rh-HRP complex by switching to positive current pulses. The extent of dye filling is monitored through the microscope using a filter combination appropriate for the dye. After fixation, standard histochemical procedures reveal HRP stained GCs in wholemount views for light microscopical examination. Furthermore, the preservation of the labeled cells and the neuropil is of a quality to allow electron microscopic analysis for synaptic input. This technique can be used in combination with LY backfilling of GCs from the optic nerve and with retinas in which GCs have been prelabeled with rhodamine beads retrogradely transported from the optic rectum as well. Introduction In this paper we describe a double labeling technique for staining single ganglion cells in the turtle retina. The technique is an extension of the fluorescent intracellular staining methods used by Tauchi and Masland (1984), Vaney (1986) and Watanabe and Rodieck (1989) or intracellular HRP-staining of ganglion cells prelabeled with rhodamine beads from the brain (Brenning and Rodieck, 1986; Bi~hl and Dann, 1988; Peichl et al., Correspondence: Dr. Helga Kolb, Ph. D., Department of Physi- ology, School of Medicine, University of Utah, 410 Chipeta Way, Research Park, Salt Lake City, UT 84108, U.S.A. 1987). The technique, as presently developed, is relatively simple, does not need prelabeling with neurotransmitters or rhodamine, but can of course also use the latter techniques, and provides a high yield of well-stained cells which we are able to examine by electron microscopy. Furthermore, be- cause the staining of individual cells is performed on the living retina, rather than on fixed retina as has been used by some of the above mentioned investigators, other labeling methods, such as im- munocytochemistry or autoradiography for puta- tive neurotransmitter candidates, can be combined with the single ganglion cell staining. Eventually it may be possible to perform intracellular record- ings in combination with the staining techniques described herein. 0165-0270/90/$03.50 ¢~ 1990 Elsevier Science Publishers B.V. (Biomedical Division)