Application of rapid-frozen substitution fixation method to immunohistochemistry

快速冷冻替代技术是研究处于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，将这项技术应用于免疫电子显微镜仍然存在一些困难。在本研究中，我们试图克服这些困难，在保存完好的超微结构中进行免疫标记。冷冻的大鼠腮腺和外分泌胰腺组织块通过在液氦预冷的铜块表面上涂抹来淬灭。组织块在以下任一介质中冷冻替换：丙酮中的臭氧、丙酮中的聚甲醛和丙酮中的戊二醛。用我们的新型蒸馏装置制备了丙酮中的多聚甲醛和丙酮中的戊二醛。替换后，嵌入修剪后的材料。超薄切片用兔抗大鼠淀粉酶抗体和蛋白A/胶体金进行免疫组织化学染色，用丙酮中的戊二醛或丙酮中的多聚甲醛冷冻取代固定的材料，包埋在Araldite 6005中，获得高标记密度。在丙酮中以Os04取代的材料，包埋在环氧树脂混合物中，并用过碘酸钠溶液蚀刻，标记密度比前者低。在Lowicryl K4M包埋材料中，标记是目前检测到的材料中最高的。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和前瞻性的信息。

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.