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

Retaining the ultrastructural arrangement of a mixed-cell culture on a solid support while processing for immunocytochemical study is a tech nical challenge. We developed a technique to study the axonal transpor t of the Herpes simplex virus from dorsal root ganglia sensory neurone s 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 inoc ulated selectively with virus. The whole preparation, retained on the cover slip, was fixed with formaldehyde 4% (freshly prepared from para formaldehyde)/glutaraldehyde 0.1%, processed by freeze substitution, a nd embedded in Lowicryl HM20 resin. The edges of the cover slip in the block were trimmed, allowing clean and complete separation from the r esin block, which retained the tissue. The resin block was placed in f resh HM20 and repolymerized. The polymerizing resin bonded strongly to the existing block, After trimming, serial sections were easily obtai ned and successfully immunolabelled for viral proteins. This is a conv enient technique for immunolabelling tissue grown on cover slips in wh ich the preservation of the ultrastructural interactions between diffe rent cells is important. It should be adaptable to a number of cell-cu lture applications and has a number of advantages over other technique s.