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A simple freeze-substitution method for electron microscopy

Authors
Journal
Journal of Ultrastructure Research
0022-5320
Publisher
Elsevier
Publication Date
Volume
15
Identifiers
DOI: 10.1016/s0022-5320(66)80119-3

Abstract

A method has been developed for freeze-substitution fixation of thin specimens. A special device shoots the specimen into propane cooled by liquid nitrogen. The substitution liquid (1.5 ml absolute methanol containing 20% uranyl acetate and 0.5 ml acrolein) is frozen in a tube below propane. After freezing of the specimen, the tube is transferred into a dry-ice acetone bath, where the substitution liquid thaws and the specimen sinks into it. Propane is then removed by suction and the preparation is kept in dry ice for 2 days and in a refrigerator for 2 more days. The combination of substituent and fixative preserves cellular structures without distortions. Neurospora hyphae thus fixed show regularly shaped mitochondria, vacuoles, and nuclei. Mitochondria are surrounded by a layer of ribosomes. Salivary glands of Drosophila show extensive ergastoplasmic lamellae with ribosomes of diffuse appearance. Older glands have amorphous looking droplets of secretion product, reduced ergastoplasm, and numerous mitochondria. Preparations can be stained with lead citrate to increase the contrast.

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