HIGH-PRESSURE FREEZING AND FREEZE-SUBSTITUTION OF NATIVE RAT-BRAIN - SUITABILITY FOR PRESERVATION AND IMMUNOELECTRON MICROSCOPIC LOCALIZATION OF MYELIN GLYCOLIPIDS

Galactocerebroside (GalC) and sulfatide are major constituent lipids i n vertebrate myelin, Their precise immunolocalization in electron micr oscopy so far has been hampered by the fact that lipids are not immobi lized by chemical fixation and thus get extracted during dehydration w ith organic solvents, Here, we examined the suitability of cryotechniq ues for the preservation and immunohistochemical localization of myeli n glycolipids in rat brain at the ultrastructural level. Native cerebr al cortex tissue, obtained by fine-needle biopsy, was cryoimmobilized by high-pressure freezing and dehydrated by freeze-substitution before embedding in Epon, This procedure resulted in an excellent preservati on of brain ultrastructure. Concomitantly, immunogold labeling of ultr athin sections with the well-defined monoclonal antibodies (mAbs) O1, O4, and R-mAb, which were shown to react with GalC and/or sulfatide an d some structurally related glycolipids, revealed a good conservation of relevant epitopes, These data suggest that in adult rat cerebral co rtex, the most relevant antigens recognized by R-mAb, O1, and O4, name ly GalC and sulfatide, are exclusively expressed in myelin structures, Because these mAbs are common markers for the identification of devel oping oligodendrocytes, this ''postembedding glycolipid-labeling techn ique'' holds great potential for studying oligodendroglial differentia tion in normal and pathological conditions at the ultrastructural leve l. (C) 1998 Wiley-Liss, Inc.