Immunocytochemical Localization of Po Protein in Golgi Complex Membranes and Myelin of Developing Rat Schwann Cells

Po protein, the dominant protein in peripheral nervous system myelin, was studied immunocytochemically in both developing and mature Schwann cells. Trigeminal and sciatic nerves from newborn, 7-d, and adult rats were processed for transmission electron microscopy . Alternating 1-lam-thick Epon sections were stained with paraphenylenediamine (PD) or with Po antiserum according to the peroxidase-antiperoxidase method . To localize Po in Schwann cell cytoplasm and myelin membranes, the distribution of immunostaining observed in 1-ltm sections was mapped on electron micrographs of identical areas found in adjacent thin sections . The first Po staining was observed around axons and/or in cytoplasm of Schwann cells that had established a 1 :1 relationship with axons. In newborn nerves, staining of newly formed myelin sheaths was detected more readily with PO antiserum than with PD . Myelin sheaths with as few as three lamellae could be identified with the light microscope . Very thin sheaths often stained less intensely and part of their circumference frequently was unstained. Schmidt-Lanterman clefts found in more mature sheaths also were unstained. As myelination progressed, intensely stained myelin rings became much more numerous and, in adult nerves, all sheaths were intensely and uniformly stained. Particulate P0 staining also was observed in juxtanuclear areas of Schwann cell cytoplasm. It was most prominent during development, then decreased, but still was detected in adult nerves. The cytoplasmic areas stained by Po antiserum were rich in Golgi complex membranes. Peripheral nervous system (PNS) myelin is synthesized by Schwann cells, and one of its major constituents is Po protein . The Po protein is an integral membrane glycoprotein (30,000 mol wt) which comprises ^-50% of the total myelin proteins in mammalian peripheral nerves (10) . We studied its localization immunocytochemically in 20-lam-thick vibratome sections and found that Po antiserum stained all peripheral myelin sheaths intensely and uniformly (25) . Similar results have been obtained using paraffm (26) and frozen (5) sections, but none of the methods used achieved cellular preservation adequate for a more detailed analysis of P o localization. Recently, Baskin et al. (2, 3) described a method to immunostain osmium-fixed, epoxy embedded tissue according to the peroxidase-antiperoxidase (PAP [20]) method . We have used this postembedding Immunocytochemical technique to detect THE JOURNAL OF CELL BIOLOGY " VOLUME 90 JULY 1981 1-6 ©The Rockefeller University Press " 0021-9525/81/07/0001/06 $1 .00 the presence of Po in newly formed myelin sheaths and in perinuclear Schwann cell cytoplasm . We also show, by tracing the distribution ofimmunostaining on electron micrographs of adjacent thin sections, that Po is in cytoplasmic areas rich in Golgi complex membranes . A preliminary report of this work has already appeared (27) . MATERIALS AND METHODS