Ultrastructural studies and immunolocalization of enamel proteins in rodent secretory stage ameloblasts processed by various cryofixation methods

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Abstract
Background: Cryofixation rapidly immobilizes cell and tissue components in their native state, thereby resulting in an ultrastructural preservation very close to the living situation. We have applied this approach to examine the morphology of secretory stage ameloblasts and the distribution of enamel proteins in these cells. Methods: Molar and incisor tooth germs from newborn mice and/or rats were quickly dissected and divided into segments. The segments were then rapidly frozen using slam, plunge or pressure freezing, freeze‐substituted and embedded in Epon. In addition, incisors from older rats were chemically fixed by vascular perfusion and also dehydrated by freeze‐substitution. Results: Well‐preserved ameloblasts were obtained with all four tissue processing methods. However, slam freezing often showed mechanical damage to the ameloblasts, particularly at the level of the distal portion of Tomes' processes which appeared severed or distorted. Plunging into liquid nitrogen‐cooled liquid propane resulted in comparatively less tissue distortion. High pressure freezing gave a relatively higher yield of well‐preserved specimens, although displacement of organelles in ameloblasts was sometimes observed, probably resulting from hydrostatic pressure. Minimal ice crystal and mechanical damage was observed in chemically fixed tooth samples processed by freeze‐substitution since such specimens are cryoprotected and their examination is not restricted to a surface layer. With all of the above cryopreparation methods, the ultrastructure of well preserved ameloblasts was, in general, similar to that obtained following conventional chemical fixation, and immunocytochemistry with an anti‐amelogenin antibody indicated no profound differences in the distribution of enamel proteins. Conclusions: These results indicate that, despite some limitations, it is possible to adequately cryofix tooth organs while preserving the architecture of ameloblasts and permitting immunolocalization of enamel proteins. Furthermore, they confirm the general morphology of secretory stage ameloblasts as currently derived from conventional chemical tissue processing.

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