Membrane stains as an objective means to distinguish isolated inner and outer hair cells

The use of isolated cochlear outer and inner hair cells has become widespread. While the morphological features of these two cell types in general are sufficiently different to allow discrimination, there are situations where confusion can arise. Small outer hair cells, particularly when they are swollen or distorted, can take on an appearance suggestive of inner hair cells. We describe here two fluorescent membrane stains, 3,3'-dihexyloxacarbocyanine iodide and rhodamine B hexyl ester, as an objective means to distinguish between cochlear hair cell types. Both stains mark the subsurface cisternae of outer hair cells thereby delineating the cell outline, and the interior of the cell shows discrete structure. On the other hand, in inner hair cells, the outline of the cell is not resolved while the interior is diffusely fluorescent. Since the two probes have different excitation and emission wavelengths (fluorescein- and rhodamine-like, respectively), this staining procedure can even be used in the presence of another fluorescent marker (for example, a calcium-indicating dye) by appropriate choice of the membrane stain.