Elemental and cytochemical localization of calcium in rat cheek epithelium

Energy-dispersive X-ray microanalysis, combined with scanning electron microscopy, was applied to 18 microns thick freeze-dried cryosections to determine the level of calcium in successive layers of the epithelium. This indicated low levels of calcium in basal cells, high in spinous cells, moderate in granular cells, lowest in the inner keratin and highest in the outer keratin layers. The potassium pyroantimonate technique was used to study the cytochemical distribution of Ca2+. The reaction product, calcium pyroantimonate (Ca-PA), was generated from cellular and intercellular calcium by perfusion of the anaesthetized rat with a solution of potassium pyroantimonate in glutaraldehyde. Ca-PA was localized in nuclei, mitochondria, lysosomal-type bodies and membrane-coating granules. A denser Ca-PA distribution was found between upper spinous and lower granular cells. The proposed identification of Ca2+ as a major component in Ca-PA was confirmed by EDTA decalcification and X-ray microprobe analysis of the reaction product. Thus X-ray microanalysis in combination with cytochemistry can localize Ca2+ in a growing and differentiating tissue such as stratified squamous epithelium.