Cytoplasmic structure in organotypic cultures of rat hippocampus prepared by rapid freezing and freeze‐substitution fixation

Synapse - Tập 13 Số 3 - Trang 195-205 - 1993
Lucas Pozzo‐Miller1,2, Dennis M.D. Landis2
1Department of Neurosciences, Roche Institute of Molecular Biology, 340 Kingsland Street, Nutley, NJ 07110‐1199
2Departments of Neurology and Neurosciences, School of Medicine, Case Western Reserve University, Cleveland, OH 44106

Tóm tắt

AbstractWe have compared rapid freezing followed by freeze‐substitution fixation with conventional aldehyde fixation as preparative methods for the electron microscopic study of organotypic cultures of neonatal rat hippocampus. Rapid freezing by contact with a copper block chilled by liquid helium was accomplished without mechanical distortion of superficial structures, and preserved structure to a depth of at least 20 μM without visible ice crystals. Freeze‐substitution fixation in acetone/osmium tetroxide, followed by en bloc staining with tannic acid and uranyl acetate, provided satisfactory staining of cytoplasm and organelles. While both preparative techniques yielded generally satisfactory results, rapid freezing provided much better preservation of astrocytic lysosomal inclusions, and afforded new views of intermediate filament substructure. Rapid freezing and freeze‐substitution fixation seemed especially well suited to the preservation of short filamentous proteins, such as those forming the membrane cytoskeleton of dendritic spines or those associated with synaptic vesicles. The combination of rapid freezing methods and organotypic culture provides an opportunity to examine cytoplasmic structure in tissue from deep regions of the brain which had previously been inaccessible to rapid freezing techniques. © 1993 Wiley‐Liss, Inc.

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Tài liệu tham khảo

10.1016/0006-8993(67)90128-X

10.1016/0006-8993(74)90463-6

10.1126/science.154.3756.1575

10.1083/jcb.102.4.1510

10.1083/jcb.99.5.1655

10.1016/0165-0270(81)90003-0

10.1016/0306-4522(84)90192-1

10.1007/BF01947561

10.1016/0166-2236(88)90007-0

10.1083/jcb.88.3.564

10.1101/SQB.1976.040.01.004

10.1083/jcb.81.2.275

10.1083/jcb.108.1.111

10.1083/jcb.88.3.660

10.1083/jcb.97.4.1169

10.1007/BF01187234

10.1002/syn.890010607

10.1016/0896-6273(88)90140-7

10.1073/pnas.82.9.3035

10.1113/jphysiol.1991.sp018549

Pozzo Miller L. D., 1991, Organization and structure of astrocytes in organotypic cultures of rat hippocampus, Soc. Neurosci. Abs., 17, 1145A

Pozzo Miller L. D., 1992, Neuronal cell death in organotypic cultures of rat hippocampus is prevented by glutamate receptor antagonists, Soc. Neurosci. Abs., 18, 440A

10.1002/ar.1091490307

10.1002/ar.1091820311

10.1083/jcb.25.1.117

10.1016/S0040-8166(70)80002-7

10.1002/cne.902280310

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Synapse

Tập 13 Số 3

195-205

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