Application of rapid-frozen substitution fixation method to immunohistochemistry

快速冷冻替代技术是研究更接近生命状态的细胞超微结构特征的一种非常有用的工具。然而，在将该技术应用于免疫电子显微镜方面仍然存在一些困难。在本研究中，我们试图克服这些困难，在保存良好的超微结构进行免疫标记。冷冻的大鼠腮腺和胰腺外分泌组织块通过在用液氦预冷的铜块表面上捣碎来淬灭。将组织块冷冻置换在以下任一种培养基中：锇的丙酮溶液、多聚甲醛的丙酮溶液和戊二醛的丙酮溶液。用新的蒸馏装置制备了多聚甲醛丙酮溶液和戊二醛丙酮溶液。替换后，将修剪的材料包埋。超薄切片用兔抗大鼠淀粉酶抗体和蛋白A/胶体金免疫染色，用戊二醛丙酮溶液或多聚甲醛丙酮溶液冷冻置换固定，Araldite 6005包埋，获得高标记密度。在用OsO_4的丙酮溶液取代的材料中，包埋在环氧树脂混合物中，用偏高碘酸钠溶液蚀刻，标记密度低于前者。Lowicryl K4 M包埋材料的标记是目前所研究的材料中最强的，本研究结果为免疫细胞化学在快速冷冻替代固定组织中的应用提供了有用的信息。

A rapid-freezing substitution technique is a quite useful tool to study the ultrastructural features of cells in a more life-like state. However, there remained several difficulties in applying the technique to immunoelectron microscopy. In this study, we tried to overcome those difficulties to perform immunolabelling in well- preserved ultrastructure.Frozen rat parotid gland and exocrine pancreas tissue blocks were quenched by smaching against a copper block surface which was pre-cooled with liquid helium. The tissue blocks were freeze-substituted in either of the following media; osmium in acetone, paraformaldelhyde in acetone, and glutaraldehyde in acetone. Paraformaldehyde in acetone and glutaraldehyde in acetone were prepared with our new distillation device. After substitution, trimmed materials were embedded. Ultrathin sections were immunostained with rabbit anti-rat-amylase antibodies and protein A/colloidal gold.High labelling density was obtained from the materials which were fixed by freeze- substitution with glutaraldehyde in acetone or paraformaldehyde in acetone, embedded in Araldite 6005. In materials substituted with Os04 in acetone, embedded in an epoxy resin mixture and etched with sodium metaperiodate solution, labelling density was lowr than the former. In Lowicryl K4M-embedded materials labelling was the ghighest among the mataerials examined so far.The results in this study present an useful and preactical information in the application of immunocytochemistry to rapid-frozen substitution fixed tissues.