Dehydration of Macroscopic Specimens by Freeze Substitution in Acetone

Freeze-substitution is based on the same principle as cryosubstitution, a dehydration procedure used in histochemistry and electron microscopy (3). Tissue to be dehydrated is removed from the fixative and placed in25°C acetone. Water within the specimen freezes immediately upon immersion, providing an internal scaffold that tends to hold its shape and dimension. Slow substitution of ice by acetone then results in only minor shrinkage. Crystal formation will distort microscopic morphology, however, if the specimen is larger than 2 mm.