Easy and reliable double-immunogold labelling of herpes simplex virus type-1 infected cells using primary monoclonal antibodies and studied by cryosection electron microscopy

Cell biology concerns the interactions between different cellular compartme nts and between the cell and the environment. The mechanisms of herpes simp lex virus type-1 (HSV-1) envelopment and the transport of virus particles a nd HSV-1 glycoproteins have not been completely investigated. It is of inte rest to examine the formation of complete virus particles and the cellular distribution of viral glycoproteins correlated with microtubules. The illus tration of these conditions by immunocytochemistry is best done by multiple labelling techniques in the same cell. Single-staining of neighbouring ser ial sections or two-face double-immunolabelling methods are not technically compatible with ultrathin cryosections. The results are reported here of a simultaneous, simple and reliable immunogold double-staining technique usi ng primary antibodies of the same species in ultrathin cryosections. Compar ed to other inactivation procedures, phosphate-buffered 3% paraformaldehyde plus 2% glutaraldehyde for 2 h at room temperature is an excellent and gen tle method to destroy free anti-IgG binding sites on the antibodies and to prevent cross-labelling, which has proven necessary for obtaining reproduci ble results on cellular distribution of tubulin and viral glycoproteins gD- 1 and gC-1.