The long range goal of this project is to combine immunocytochemical methods with light and electron microscopy to study cellular mechanisms on myelin formation, breakdown, and regeneration. Current studies and major findings are: (1) Tests of postembedding light and electron microscopic immunocytochemical methods to evaluate: (a) effects of epon section pretreatments on tissue fine structure and on distributions of herpes virus type 2 (HSV-2) immunoreactivity in sections of cultured cells infected with this virus. In H202 pretreated semithin and thin sections, cellular structure is well preserved and reaction product is located on HSV-2 virions after use of either the peroxidase-antiperoxidase (PAP) or the biotin-avidin immunostaining procedure. Use of sodium ethoxide in concentrations that removed significant amounts of epon during pretreatment also severely altered cell and organelle fine structure; therefore, this pretreatment agent could not be used to study distribution of immunoreactivity electron microscopically. (b) Usefulness of Lowicryl as an embedding medium for light and electron miscroscopic localization of the Po myelin glycoprotein in developing PNS tissue. Preliminary results indicated that it can be demonstrated in aldehyde-fixed PNS without section pretreatment. If confirmed and extended to thin sections examined electron microscopically, this embedment will be tested extensively for use in nucleic acid hydridization studies. (2) Immunocytochemical comparison of P2 and P1 basic protein expression in Schwann cells of developing rat VI nerves. Immunoreactivity was first detected on day 2 and the percentage of myelinated fibers immunostained rose rapidly thereafter, P1 greater than P2, so that by day 20, 80% were P2 positive. When measured quantitatively, straining intensities were not uniform in all myelin sheaths. Intensity variation was greater with P2 than with P1; it decreased with increasing age.
