Immunocytochemical localization of basic protein in major dense line regions of central and peripheral myelin

To localize basic protein (BP) in the lamellar structure of central and peripheral myelin, we perfused newborn and 7-11-day rat pups with a phosphate-buffered fixative that contained 4% paraformaldehyde and 0.05 or 0.2% glutaraldehyde. Teased, longitudinally split or "brush" preparations of optic and trigeminal nerves were made by gently teasing apart groups of myelinated fibers with fine forceps or needles. Some of these preparations were immunostained without pretreatment in phosphate- buffered antiserum to BP according to the peroxidase-antiperoxidase method. Others were pretreated in ethanol before immunostaining. Then, all of them were dehydrated, embedded in Epon, and sectioned for electron microscopic study. In optic and trigeminal nerves that were not pretreated, myelin, glial cells, and their organelles were well preserved. BP immunostaining was present on cytoplasmic faces of oligodendroglial and Schwann cell membranes that formed mesaxons and loose myelin spirals. In compact central and peripheral myelin, reaction product was located in major dense line regions, and the myelin periodicity was the same as that observed in unstained control myelin that had been treated with preimmune serum. In ethanol- pretreated tissue, the myelin periodicity was reduced but dense line staining still was present. Our immunocytochemical demonstration of dense line localization of BP in both CNS and PNS myelin that was not disrupted or pretreated with solvents is important because of conflicting evidence in earlier immunostaining studies. Our results also support biochemical and histochemical evidence suggesting that BP exists in vivo as a membrane protein interacting with lipids on the cytoplasmic side of the bilayer in the spirally wrapped compact myelin membrane.