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Freeze-thawing as a preparatory technique for electron microscopy

Journal of Ultrastructure Research
Publication Date
DOI: 10.1016/s0022-5320(62)80034-3


Small pieces of mouse kidney cortex were frozen by immersion in isopentane cooled to −160°C by liquid nitrogen. The tissue was warmed to −18°C and 10- μ sections were cut in a cryostat. Thawing was effected by immersion in buffered osmium tetroxide at +4°C. Fixation and embedding in methacrylate or Epon 812 followed. Ultrathin lead stained sections showed few vacuoles due to ice crystals. The probable sites of intra-cytoplasmic ice crystals were characterized by areas lacking fine structure and slightly denser than the surroundings. Brush border, mitochondria, cytoplasmic membranes, and ribosomes were well preserved except for mitochondrial swelling. Blebbing of the outer nuclear membrane and vacuolization of the chromatin were seen. Slow cooling at 2°C/minute to −18°C followed by section cutting and fixation as above produced ultrathin sections showing fine structure preservation nearly equivalent to that seen after fast freezing, except that intracellular ice crystal sites were not seen.

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