A method for selective intracellular labeling of immunostained neurons in turtle retina

We describe a method for direct intracellular staining under visual control of immunolabeled neurons in the turtle retina. Substance P was the antiserum used. It labels two different sizes of ganglion cells in turtle retina. Intracellular labeling under visual control was achieved by iontophoresis of Lucifer yellow or Neurobiotin. The best immunolabeling of substance P-immunoreactive (SP-IR) ganglion cells occurred after either Triton X-100 or freeze-thaw techniques to get good penetration of the antisera. However, this inevitably resulted in leaky cells and inadequate morphology of the ganglion cells subsequently stained by Lucifer yellow and Neurobiotin. Most successful immunocytochemical labeling followed by intracellular labeling was achieved with light fixation (15 min in 4% paraformaldehyde) and long incubation time in the primary antiserum (4 days). Before intracellular labeling, dendritic tree shape, dendritic field size, and stratification of SP-IR ganglion cells were not sufficiently revealed for correct classification of these cells. After the selective intracellular staining described here, we were able to identify and characterize one of the populations of substance P-IR ganglion cells types as large-field, monostratified G20 ganglion cells.