Abstract

In situ hybridization (ISH) is a standard method for localizing DNA or RNA sequences in tissue or cell preperation. The technique was developed at the electron-microscopic level, and enables the precise subcellular localization. A method was developed for detection of specific viral DNA. We have tested various methods and technique to detect specific viral DNA through ISH at the electron microscopic level. Postembedding method of ultrastructural ISH was developed and successfully applied for the detection of human papillomavirus type 16 in squamous cell carcinoma of the uterine cervix and Epstein-Barr virus in EBV-infected leukemia cell line. The following results are made. The best results were obtained using 0.2% glutaraldehyde and 4% paraformaldehyde fixed tissue or cell block. The labelling was best observed on Unicryl resin and Lowicryl K4M resin sections. Epon sections showed no reactivity. Thin sections of Unicryl resin were more easier than Lowicryl K4M resin. Enzymatic predigestion with proteinase K, pepsin and trypsind gave good results. However, high concentration of these produce poor results due to excessive destruction of the cellular components. Alkali treatment with 0.5N sodium hydroxide produced successful results in denaturation of target DNA. The labelling density of gold particles was independent of incubation time or temperature in hybridization step. The viral DNA labelling was localized mainly within the nucleus, both within and at the edge of electron dense regions, and below the nuclear membrane. And the labelling was seen in the form of a dense, roughly spherical shape. In conclusion, the best results are obtained by the conditions that tissue fixed by 0.2% glutaraldehyde and 4% paraformaldehyde solution, embedded with Unicryl resin, protein denaturation by 0.1ul/ml proteinase K, DNA denaturation by 0.5N sodium hydroxide, and reaction with DNA probe.

• Keywords: Ultrastructure;In situ hybridization