Cryofixation, cryosubstitution, and immunoelectron microscopy: potential role in diagnostic pathology

In immunoelectron microscopic investigations, retention of antigenic sites is crucial. Methods for preparing samples for conventional electron microscopy involve chemical fixatives followed by dehydration in organic solvents and embedding in plastic resins, all procedures potentially detrimental to antigenicity. Cryomethods provide a physical fixation alternative for the preparation of biological samples for ultrastructural, immunocytochemical, and microanalysis studies without the use of any chemicals. This can be particularly useful in diagnostic pathology, providing an alternative to conventional fixation methods which sometimes destroy the antigen in question. The recent development of a portable cryofixation device, the PS1000 Portable Metal Mirror Ultra-Rapid Cryofixation Unit (Delaware Diamond Knives, Inc., Wilmington, DE, USA), provides an opportunity to freeze tissue immediately after procurement for use in diagnostic immunocytochemistry studies. This feasibility study examined the quality of tissue preservation with this device, in terms of both preservation of cellular ultrastructure and immunolabeling. Human tonsil and thymus tissue was slam frozen and, after cryosubstitution in Lowicryl K11M, was examined by immunoelectron microscopy. Good ultrastructural preservation was obtained and reasonable immunolabeling with antibodies to AE1/AE3 keratin filaments was also observed.