Cryo‐section immunolabelling of difficult to preserve specimens: advantages of cryofixation, freeze‐substitution and rehydration

Abstract

Electron microscopic immunolabelling of ultrathin thawed cryo-sections, according to the method of Tokuyasu, is widely used as a very sensitive high-resolution localization technique. Its main advantages are that antigens remain in a hydrated environment prior to immunolabelling, and that antigen accessibility is improved compared with resin section labelling. However, the quality of structural appearance and antigenicity depends highly on the limitations of the initial conventional chemical fixation step, such as slow diffusion and selective reaction/cross-linking of fixative molecules. Cryofixation, instead of conventional chemical fixation, followed by freeze-substitution/chemical fixation, rehydration and further processing for Tokuyasu cryo-sectioning leads to an improved preservation of both ultrastructure and antigenicity. This is especially true for tissues which are difficult to preserve by conventional chemical fixation at ambient temperatures, such as plant material, Drosophila embryos or nematode tissue. In particular labile and highly dynamic structures (for example, microtubules and Golgi apparatus) are remarkably better preserved. These improvements are also valid for light microscopic applications.