THE SECTION FREEZE-SUBSTITUTION TECHNIQUE: I. METHOD

A new technique of freeze-substitution, called "Section Freeze-Substitution," is described. Thin pieces of tissue are quick-frozen as for freeze-drying, and sectioned in a cryostat. The free sections are kept in dry ice-temperature absolute acetone overnight, then picked up with cover glasses, coated with celloidin if necessary, and air dried. The sections adherent to the cover glasses can be stained or incubated in substrate solution directly at room temperature (23-27°C). Finished slides can be made front fresh tissues [See figure in the PDF file] within 24 hours. With few exceptions, both oxidative and hydrolytic enzymes, soluble isotopes, and other chemical substances can be precisely demonstrated in a single substitution process. The cytology of the frozen-substituted tissue is excellent. No other histochemical tissue processing method preserves so much of a particular enzyme. The procedure is simple and the results are highly reproducible.