Takemori N, Saito N, Hirai K, Onodera R, Miyakawa K, Namiki M
Third Department of Internal Medicine, Asahikawa Medical College, Japan.
Hokkaido Igaku Zasshi. 1994 Mar;69(2):249-57.
Cryoultramicrotomy applying immunogold staining (immunocryoultramicrotomy) is a useful method to demonstrate the localization of antigens in biological specimens. Recently, we have developed a modified method for immunocryoultramicrotomy using gelatin as an embedding medium (gelatin embedding method). In this study, we compared the results obtained by the gelatin embedding method with those obtained by a conventional method applying polyvinyl alcohol embedding (PVA embedding method). Both methods were easy to perform, and yielded fairly satisfactory results in terms of detecting antigens and preserving ultrastructures. According to the gelatin embedding method, membranes enclosing organelles revealed a negatively stained image, and organelles were clearly delineated by an electron-lucent layer. In contrast, a positively stained image was obtained by the PVA embedding method; however, the delineation of organelles was somewhat inferior to that of the gelatin embedding method. Although these methods were useful to detect various biological antigens, they had some limitations in sensitivity. Prior glutaraldehyde fixation was requisite for cryosectioning and preserving ultrastructures, but it caused the loss of antigenicity to some extent. This should be taken into account when evaluating the results obtained by immunocryoultramicrotomy. In this paper, we present the detailed procedures of the gelatin embedding method, as well as the PVA embedding method, and demonstrate the localization of myeloperoxidase, lysozyme and CD3 molecules in human blood cells using both methods.
应用免疫金染色的冷冻超薄切片术（免疫冷冻超薄切片术）是一种用于在生物标本中显示抗原定位的有用方法。最近，我们开发了一种以明胶为包埋介质的免疫冷冻超薄切片术改良方法（明胶包埋法）。在本研究中，我们将明胶包埋法得到的结果与应用聚乙烯醇包埋的传统方法（PVA包埋法）得到的结果进行了比较。两种方法都易于操作，在检测抗原和保存超微结构方面都产生了相当令人满意的结果。根据明胶包埋法，包裹细胞器的膜呈现负染图像，细胞器由电子透明层清晰勾勒。相比之下，PVA包埋法获得的是正染图像；然而，细胞器的勾勒在某种程度上不如明胶包埋法。虽然这些方法对于检测各种生物抗原很有用，但它们在灵敏度方面存在一些局限性。冷冻切片和保存超微结构之前需要进行戊二醛固定，但这在一定程度上导致了抗原性的丧失。在评估免疫冷冻超薄切片术获得的结果时应考虑到这一点。在本文中，我们介绍了明胶包埋法以及PVA包埋法的详细步骤，并使用这两种方法展示了人血细胞中髓过氧化物酶、溶菌酶和CD3分子的定位。
