A technique of decalcification suited to electron microscopy of tissues closely associated with bone

Systematic experiments have resulted in a method of decalcification which, although specifically developed for cytological study of reptilian aural tissue, should be applicable to other problems involving electron microscopy of organic components of bone or tissues closely associated with bone. Reptilian otic capsules and control specimens of mammalian hepatic tissue were subjected to planned variations of fixatives and decalcifying agents, embedded, sectioned, and evaluated by light and electron microscopy. Additionally, applicability of the more satisfactory agents to other mammalian tissue was checked by limited testing on specimens from Mongolian gerbils. Acceptable results were achieved in tissues fixed in glutaraldehyde, and then decalcified in cold 0.1 M solutions of disodium or tetrasodium EDTA (adjusted to pH 7.2–7.4 with sodium hydroxide or versenic acid, respectively), containing 4.0% glutaraldehyde; following decalcification, specimens were washed in buffer, postfixed in osmium tetroxide, and embedded in epoxy resins with minor modification of standard techniques. Acceptable cytological detail and morphological relationships were retained, and extended treatment with the decalcifying solution did not apparently result in degradation of tissues.