Ultrastructural localization of S100A3, a cysteine-rich, calcium binding protein, in human scalp hair shafts revealed by rapid-freezing immunocytochemistry

We have characterized the subcellular distribution of S100A3, a cysteine-ri ch calcium binding protein, in human scalp hair shaft. This was accomplishe d using rapid-freezing immunocytochemistry, a technique that combines rapid -freezing, freeze-substitution fixation without chemical fixatives, and sub sequent electron microscopic detection of immunocytochemical labeling. This technique preserves both the antigenicity and the ultrastructural integrit y of fully keratinized tissues, which are highly unmanageable when prepared for immunoelectron microscopy. In the hair shaft, S100A3 was primarily ide ntified in the endocuticle and was also present in the intermacrofibrillar matrix surrounding macrofibril bundles of intermediate filament keratins in cortex cells. Double immunolabeling of S100A3 and hair keratins revealed t he in situ spatial relationship between them. In the endocuticle, S100A3 wa s present on the inner portion of the endocuticle adjacent to the cell memb rane complex, whereas hair keratins were present on the outer portion. Thes e results provide the first ultrastructural evidence that an S100 protein i s localized in specific subcompartments in human hair cells.