Preparing Hydra for Freeze-Fracture and Freeze-Etching
The freeze-fracture technique is particularly advantageous for the study of cell surfaces and intercellular junctions. In general, it involves the rapid freezing of tissues, either with or without partial sublimation of ice (etching). By evaporating a film of pure carbon on the exposed surface, a replica of the cell surface is obtained which reveals its intimate contours. Contrast is obtained by shadowing the exposed surface with evaporated platinum prior to forming the replica. The tissue is removed from the replica by digestion in caustic solutions prior to recovery of the replicas for viewing at the electron microscope (Koehler, 1972). When a fracture occurs along the plane of a biological membrane it tends to split the lipid core and reveal unique surfaces for subsequent replication (Branton, 1966). Junctional regions can be identified by the arrangement of intramembranous particles, which appear to consist predominantly of protein. The distribution of surface components can be localized by combining the freeze-etching procedure with surface probes conjugated with large molecular weight compounds such as ferrittn.
KeywordsExposed Surface Sodium Cacodylate Lipid Core Rapid Freezing Caustic Solution
Unable to display preview. Download preview PDF.
- Koehler, J. 1972. The freeze-etching technique. In: Principles and Techniques of Electron Microscopy. Biological Applications, vol. 2 ( M. Hayat, ed.), Van Nostrand Reinhold, New York, pp. 53–98.Google Scholar