Although the omission of chemical fixation would be most desirable, protein and lipid fixation is usually necessary for morphological studies of cells and tissues. On the other hand, application of immunoelectron microscopic techniques to freeze-substituted samples requires that the concentrations of fixatives be as low as possible. This means abandoning high quality preservation and visibility of ultrastructural details. In any case, a compromise has to be found (Robards and Sleytr, 1985; Müller, 1988).

The technique of freeze-substitution combines shock-freezing, substitution of amorphous ice by methanol, embedding in low temperature resin, polymerization of the resin in ultraviolet light at low temperature, and conventional ultra-thin sectioning. After optimization, this approach not only gives reasonable preservation of cell ultrastructure, but also protects antigenic components in the cell from severe loss of antigenicity. This is important when immunoelectron microscopy is applied.

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