A reliable method for obtaining matched replicas of freeze-fractured cell suspensions.
A method that overcomes the majority of practical problems involved in performing matched freeze-fracture replication of cell suspensions is described. Specimens are rapidly frozen between copper support plates. The prior attachment of gold grids to the plates using Formvar ensures the exact alignment of corresponding grid squares and the production of a central fissure running through the specimen upon fracturing. Grids, with replicas permanently attached, are removed from the support plates by dissolving the Formvar. Sample digestion and rinsing of replicas is by a gentle procedure employing glass capillaries as a means of handling the grids. Matched replication of plant and animal cells has been achieved by this methods. Apart from the excellent preservation of the membrane ultrastructure of unfixed cells and the capacity for matching both membrane halves this technique also enhances the fracture characteristics of certain specimens, notably isolated protoplasts, providing larger fracture faces than are obtained by the knife-splintering method.
D002462:Cell Membrane / Q000648:ultrastructure; D005614:Freeze Fracturing / Q000379:methods*; D010944:Plants / Q000648:ultrastructure; D011523:Protoplasts / Q000648:ultrastructure