Combined in situ hybridization and immunohistochemistry for automated detection of cytomegalovirus and p53

Background: Cytomegalovirus (CMV) infection has been shown to be associated with p53 overexpression in coronary artery restenosis. We investigated the occurrence of this association in other forms of CMV infection using an automated in situ hybridization (ISH) technique. Methods and Results: We performed ISH for CMV using digoxigenin-labeled or biotinylated probes followed by avidin-alkaline phosphatase and nitroblue tetrazolium color substrate. Immunohistochemistry (IHC) was then performed using an anti-p53 antibody utilizing streptavidin-immunoperoxidase and 3,3′-diaminobenzidine tetrahydrochloride as a chromogen. Sixteen cases with characteristic cytomegalic inclusions from a variety of body sites were examined. All 16 cases were positive for CMV by ISH. Nine of sixteen expressed nuclear p53. Six of these nine cases showed viral cytopathic effect in the cells with p53 expression. In an illustrative case, double colocalized staining for CMV and p53 protein was demonstrated in individual cytopathic cells. When microwave antigen retrieval was necessary, ISH was performed before IHC, and our standard microwaving time was reduced by two-thirds. Conclusions: The colocalization of p53 protein overexpression with CMV within single cells adds further evidence that this overexpression is a viral-induced phenomenon. The combined ISH and IHC assay can be carried out in a rapid automated mode, increasing the ease of investigating relationships between message and protein expression within single cells in a wide variety of settings.