The plaque-assay technique was used as a tool to determine the optimal conditions for adsorption of polyoma virions to host cells. Using these optimal conditions of adsorption, an electron microscopy study of the early events of infection was performed. By electron microscopy and autoradiography, it was demonstrated that both the viral coat proteins and DNA arrive simultaneously in the nucleus as early as 15 min postinfection. When horseradish peroxidaselabeled virions, pseudovirions, and capsids were used to infect cells, only the particles with nucleic acid or a factor(s) associated with the nucleic acid, i.e., histones, appeared to enter the nucleus. Moreover, when virions were used to infect either permissive or nonpermissive cells, identical early events of viral infection, i.e., adsorption, penetration, and nuclear transport, were observed, suggesting that these early events of infection are a property of the virion and not the host cell.

The human cervical canal is lined by three epithelial cell types: mucous, ciliated and reserve cells. The origin of immunoglobulins within the endocervix has not been clearly defined. In 1968, Pozzuoli et al presented immunoelectrophoretic and immunofluorescent evidence for the existence of IgA and IgG within both human cervical mucus and plasma cells which were present beneath the endocervical epithelium. Masson, Heremans and Ferin in 1969 analyzed pooled samples of cervical mucus by chromatography on DEAE-cellulose columns and were able to identify IgA and IgG as well as several other soluble proteins. In 1970 Lippes et al were able to identify IgA and IgG in the human cervix by immunocytochemical localization at the light microscopic level.Using a direct peroxidase conjugated antisera to human IgA and IgG, we have been able to confirm the presence of these immunoglobulins at the ultrastructural level and to localize both IgA and IgG within the granules of endocervical mucous cells.

The presence of nonspecific staining artifacts is a potential problem in ultrastructural immunocytochemistry. In the course of staining for lysozyme in the human and rabbit endocervix, the peroxidase-antiperoxidase complex (PAP) was found to bind to mucous granules in a selective and nonspecific manner. Sections etched in 10% aqueous H20z, incubated for 5 minutes in 1 5 0 PAP in Tris-buffered saline (TBS) and subsequently treated with 3, 3'-diaminobenzidine-H202, revealed a staining precipitate within the matrix of mucous granules. The same selective and nonspecific staining could also be visualized when horseradish peroxidase (1 mg/ml) was substituted for PAP in the immunocytochemical sequence. Thus, this staining method made the reliable demonstration of the desired antigen difficult. The affinity of PAP for mucous granules can be completely eliminated by subjecting sections, previously etched in H202 to expose the antigenic sites, to a 3-minute incubation in immunoglobulin (1:lO human IgA, 15 human IgG, or 1 5 antihuman IgG) immediately prior to using PAP in the standard immunocytochemical staining sequence. The use of this modification to Sternberger's unlabeled antibody-enzyme method is recommended because it allows for the elimination of all nonspecific staining artifacts without interfering with specific localization by the primary antibody. The mechanism that causes nonspecific binding of peroxidase to mucous granule constituents is unclear, although carbohydrate binding may play a role in the interaction between mucous granules and peroxidase.

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