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

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.