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Journal of Electron Microscopy 48(5): 621-628 (1999)
© 1999 Oxford University Press

Co-localization of HSV-1 DNA and ICP35 protein by in situ hybridization and immunocytochemistry

Hiroyuki Morioka1,*, Kappei Kobayashi2, Masayoshi Tachibana3 and Jiro Imanishi2

1Laboratory of Electron Microscopy
2Department of Microbiology, Kyoto Prefectural University of Medicine Kamigyo-ku, Kyoto 602–8566
3Research Institute, Saitama Cancer Center 818 Komuro, Ina, Saitama 362–0806, Japan

*To whom correspondence should be addressed: E-mail: hmorioka{at}basic.kpu-m.ac.jp

In an effort to obtain a better signal-to-noise ratio and ultrastructural preservation, we sought to improve electron microscopic in situ hybridization technique. In our method, protease treatment was omitted and visualization of the digoxigenin (DIG)-labelled deoxyribonucleic acid (DNA)-probe was enhanced using a three-step procedure. These improvements allowed us to localize viral DNA with good signal-to-noise ratio. DNA specific to herpes simplex virus 1 (HSV-1) was localized by this method to HSV-1 infected cultured cells; DNA was not observed in the empty-cored HSV- 1. Using this method and the immunogold cytochemical method, we co-localized viral DNA and capsid protein ICP35 on Lowicryl-embedded sections of HSV-1 infected cells. Interestingly, labelling for both DNA and ICP was observed on some HSV-1 particles in cell nucleus. This finding is consistent with the notion that ICP35 is necessary for assembly of viral DNA. Combination of in situ hybridization, and immunocytochemical techniques is a powerful tool for examination of the functional relationship between viral DNA and proteins and help us to study protein function in viral multiplication.

Keywords     herpes simplex virus type 1, electron microscopic in situ hybridization, dioxigenin-labelled DNA, electronmicroscopic immunogold cytochemistry, ICP35, IgG-gold

Received     18 March 1999, accepted 7 June 1999


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