We used preparative freeze-fracture to gain cytochemical access to the plasma membranes and extracellular matrices (basement membrane, mesangial matrix) of rat kidney glomeruli. In addition to its proven capacity to locate transmembrane proteins, to follow intracellular traffic of membrane proteins, and to characterize both extracellular and cytoplasmic matrices, the development of the modes of application of fracture-label led to the discovery of an elective mode for storage of tissue biopsies for future cytochemical study. The sequence that we propose, based on our fracture-label studies since 1980 involves chemical fixation, gradual impregnation in 25% (v/v) glycerol followed by rapid freezing in the liquid phase of partially solidified Freon 22 (-156 degrees C) cooled by liquid introgen. We showed that these specimens, which can be stored indefinitely in liquid introgen, can be thawed without loss of ultrastructional detail or of antigenic activity. This sequence allows for the collection of tissue biopsies over extended periods with the cytochemical labeling done in a single experiment once a sufficient number and diversity of specimens have been collected.
