Nanci A, Kawaguchi H, Kogaya Y
Department of Anatomy, Université de Montréal, QC, Canada.
Anat Rec. 1994 Apr;238(4):425-36. doi: 10.1002/ar.1092380402.
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.
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.
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.
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.
冷冻固定能迅速将细胞和组织成分固定在其天然状态，从而实现与活体状态非常接近的超微结构保存。我们已应用此方法来研究分泌期成釉细胞的形态以及这些细胞中釉质蛋白的分布。
迅速解剖新生小鼠和/或大鼠的磨牙和切牙牙胚，并将其分成节段。然后使用骤冷、投入或高压冷冻法将节段迅速冷冻，进行冷冻置换并包埋于环氧树脂中。此外，对成年大鼠的切牙进行血管灌注化学固定，并同样通过冷冻置换进行脱水处理。
所有四种组织处理方法均获得了保存良好的成釉细胞。然而，骤冷冷冻常常显示对成釉细胞有机械损伤，特别是在托姆斯突远端部分，此处似乎出现断裂或扭曲。投入液氮冷却的液态丙烷导致的组织变形相对较少。高压冷冻获得保存良好标本的产量相对较高，尽管有时观察到成釉细胞中的细胞器移位，这可能是由静水压力导致的。在通过冷冻置换处理的化学固定牙齿样本中观察到冰晶和机械损伤最小，因为此类标本经过了冷冻保护，并且其检查不限于表层。使用上述所有冷冻制备方法，保存良好的成釉细胞的超微结构总体上与传统化学固定后获得的结构相似，并且用抗釉原蛋白抗体进行的免疫细胞化学表明釉质蛋白的分布没有显著差异。
这些结果表明，尽管存在一些局限性，但在保存成釉细胞结构并允许对釉质蛋白进行免疫定位的同时，能够充分冷冻固定牙齿器官。此外，它们证实了目前从传统化学组织处理获得的分泌期成釉细胞的一般形态。
