Herpes simplex virus type 1-infected cells studied by optimized double-immunogold cryosection electron microscopy

The mechanisms of Herpes simplex virus type 1 (HSV-1) envelopment and the transport of virus particles and HSV-1 glycoproteins in the host cell are partly unknown. Intracellular transport and the formation of infectious virus particles may depend on the presence of intact microtubules. A simultaneous, simple and reliable immunogold double-staining technique using two primary antibodies of the same species in ultrathin cryosections of HSV-1 infected cultured human fibroblasts (MRC-5) is described. Phosphate buffered 3% paraformaldehyde plus 2% glutaraldehyde 2h at room temperature is superior to other inactivation procedures to destroy free anti-IgG binding sites on the antibodies and to prevent cross-labeling. This method allows reproducible results on cellular distribution of tubulin and viral glycoproteins gD-1 and gC-1.