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High‐pressure freezing and freeze‐substitution of native rat brain: Suitability for preservation and immunoelectron microscopic localization of myelin glycolipids
Author(s) -
Kirschning Eckhard,
Rutter Gabriel,
Hohenberg Heinz
Publication year - 1998
Publication title -
journal of neuroscience research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.72
H-Index - 160
eISSN - 1097-4547
pISSN - 0360-4012
DOI - 10.1002/(sici)1097-4547(19980815)53:4<465::aid-jnr8>3.0.co;2-4
Subject(s) - immunogold labelling , glycolipid , myelin , epitope , immunoelectron microscopy , ultrastructure , monoclonal antibody , biology , immunocytochemistry , galactocerebroside , biochemistry , cerebral cortex , chemistry , oligodendrocyte , microbiology and biotechnology , immunohistochemistry , antigen , anatomy , antibody , immunology , neuroscience , central nervous system , endocrinology
Galactocerebroside (GalC) and sulfatide are major constituent lipids in vertebrate myelin. Their precise immunolocalization in electron microscopy so far has been hampered by the fact that lipids are not immobilized by chemical fixation and thus get extracted during dehydration with organic solvents. Here, we examined the suitability of cryotechniques for the preservation and immunohistochemical localization of myelin glycolipids in rat brain at the ultrastructural level. Native cerebral 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 preservation of brain ultrastructure. Concomitantly, immunogold labeling of ultrathin sections with the well‐defined monoclonal antibodies (mAbs) O1, O4, and R‐mAb, which were shown to react with GalC and/or sulfatide and some structurally related glycolipids, revealed a good conservation of relevant epitopes. These data suggest that in adult rat cerebral cortex, the most relevant antigens recognized by R‐mAb, O1, and O4, namely GalC and sulfatide, are exclusively expressed in myelin structures. Because these mAbs are common markers for the identification of developing oligodendrocytes, this “postembedding glycolipid‐labeling technique” holds great potential for studying oligodendroglial differentiation in normal and pathological conditions at the ultrastructural level. J. Neurosci. Res. 53:465–474, 1998. © 1998 Wiley‐Liss, Inc.