New Techniques:The Use of Fixed-Frozen Tissue in Routine Ultrastructural Evaluation and Immunogold Labeling

Abstract

A method of freezing fixed surgical tissue specimens for long-term storage and subsequent resin embedding was developed. The method entails fixation of the specimens in Karnowsky's liquid, cryoprotecting with 10% sucrose, freezing in melting Freon gas, and storage at -80°C. When needed, even after years, the frozen tissue pieces are thawed in lukewarm (37°C) buffer and embedded in resin (Epon or Lowicryl). Ultrastructural detail as well as immunocytochemical reactivity of a wide variety of cellular constituents are well preserved. The method is practical and allows retrospective embedding in Lowicryl resin (a technique not easily applicable in everyday routine) for subsequent ultrastructural immunolocalization studies.

Key Words:

• freeze-thawing
• immunogold labeling
• long-term tissue storage
• ultrastructural preservation