The axonal transmission of Herpes simplex virus to epidermal cells: a novel use of the freeze substitution technique applied to explant cultures retained on cover slips

Abstract

Retaining the ultrastructural arrangement of a mixed-cell culture on a solid support while processing for immunocytochemical study is a technical challenge. We developed a technique to study the axonal transport of the Herpes simplex virus from dorsal root ganglia sensory neurones to epidermal cells. Autologous explants of human foetal dorsal root ganglia and skin were cultured on plastic cover slips. Axon fascicles grew from the ganglia to the epidermal cells and the ganglia were inoculated selectively with virus. The whole preparation, retained on the cover slip, was fixed with formaldehyde 4% (freshly prepared from paraformaldehyde)/glutaraldehyde 0.1%, processed by freeze substitution, and embedded in Lowicryl HM20 resin. The edges of the cover slip in the block were trimmed, allowing clean and complete separation from the resin block, which retained the tissue. The resin block was placed in fresh HM20 and repolymerized. The polymerizing resin bonded strongly to the existing block. After trimming, serial sections were easily obtained and successfully immunolabelled for viral proteins. This is a convenient technique for immunolabelling tissue grown on cover slips in which the preservation of the ultrastructural interactions between different cells is important. It should be adaptable to a number of cell-culture applications and has a number of advantages over other techniques.