Application of a platinum replica method to the study of the cytoskeleton of isolated hair cells, supporting cells and whole mounts of the organ of Corti.

We adapted a method of platinum replica to study the cytoskeleton of isolated cells of the guinea pig organ of Corti. This technique combined high image resolution with the ability to visualize the three-dimensional organization of the cytoskeleton of a whole cell. The procedure includes: isolation of hair cells and supporting cells using collagenase digestion, attachment of the cells to a coverslip, detergent extraction, chemical fixation, critical point drying, platinum/carbon coating, and transmission electron microscopy analysis. By using the method of platinum replica, we confirmed the existence of structural domains in the cortical lattice of outer hair cells. Based on the analysis of the partly destroyed cortical lattice, we propose that circumferential filaments are underlined with a thin flexible network. In addition, we established that the base of each stereocilium had a cone-like expansion of actin filaments and was surrounded by a thin bundle of filaments. We also produced replicas of the protrusion of the cuticular plate into the cytoplasm (infracuticular network) and the reticular lamina cytoskeleton. Our data indicated that the platinum replica method is useful for studying structural interactions among different cytoskeletal elements in the reticular lamina, as well as the cortex of outer hair cells and the cytoskeleton of supporting cells.