A copper block method for freezing non-cryoprotected tissue to produce ice-crystal-free regions for electron microscopy. II. Evaluation using freeze fracturing with a cryo-ultramicrotome.
Two methods are described for fracturing unfixed, uncryoprotected mouse liver that had been frozen by contact with a copper block at liquid nitrogen temperature. The best method involved precise fracturing of a few microns of surface tissue with a cooled glass knife,  using the Cryokit attachment of the LKB III ultramicrotome. The resulting replicas were free from electron microscopically-visible ice crystals. A noticeable feature of replicas from unfixed, uncryoprotected tissue was the extent of plastic deformation of certain cellular structures. Cytoplasmic macromolecules, and to a lesser extent intramembranous particles, often appeared to be 'stretched' to form fibrils.
D000818:Animals; D005614:Freeze Fracturing / Q000295:instrumentation / Q000379:methods*; D005615:Freezing; D006652:Histological Techniques / Q000295:instrumentation; D008099:Liver / Q000648:ultrastructure; D051379:Mice; D008854:Microscopy, Electron; D008867:Microtomy