Improvement of Freeze-Substitution by Programmed Rewarming

Abstract

Since freeze-substitution was introduced into histological technique by Simpson (1941) and Lison (1949), numerous authors have published variations of this method. There have been divergent opinions on the best media to use as substituents, and on the temperatures and times at and for which substitution should proceed. At desirable low temperatures, Pearse (1968) found such a slow rate of penetration that even by increasing the time of substitution “up to weeks or even months” only little change would be produced. Persijn et al. (1964) described a complicated apparatus which they used in their laboratory to solve the problem of regulating the temperatures in the process of freeze-substitution.