Goto T, Kohno T, Nakano T, Fujioka Y, Morita C, Sano K
J Electron Microsc (Tokyo). 2001;50(1):73-6. doi: 10.1093/jmicro/50.1.73.
Electron microscopic in situ hybridization (EM-ISH) is a useful method in determining the localization of a specific nucleic acid at the ultrastructural level. Since the EM-ISH protocol includes many steps, no standard protocol for EM-ISH is available yet. In this study, we optimized quantitatively the critical conditions with respect to embedding resin, nucleic acid labeling and hybridization reaction time, by using adenovirus-infected cells as the indicator cells. The optimal detection of an adenovirus-specific nucleic acid was obtained by overnight hybridization reaction on sections embedded in Lowicryl K4M resin. Random-primed-labeled probes improved the reactivity. At least 60% of virus particles in paracrystalline arrays was found to contain viral DNA. These arrays in adenovirus-infected cells are useful in evaluating quantitatively the efficiency of protocols of EM-ISH.
电子显微镜原位杂交（EM-ISH）是在超微结构水平上确定特定核酸定位的一种有用方法。由于EM-ISH方案包含许多步骤，目前尚无EM-ISH的标准方案。在本研究中，我们以腺病毒感染的细胞作为指示细胞，对包埋树脂、核酸标记和杂交反应时间等关键条件进行了定量优化。通过在Lowicryl K4M树脂包埋的切片上进行过夜杂交反应，可实现腺病毒特异性核酸的最佳检测。随机引物标记的探针提高了反应性。发现至少60%的平行排列病毒颗粒含有病毒DNA。腺病毒感染细胞中的这些排列有助于定量评估EM-ISH方案的效率。
