Abstract
Publisher Summary This chapter reviews literature on cryoimmobilization, freeze-substitution, and resin embedding for ultrastructural and immunocytochemical transmission electron microscopy (TEM) studies of plants and fungi. It illustrates the reasons behind the difficulty to fix plants and fungi usually, while they are rather easy to freeze. It mentions the favorable features of plants and fungi, which make cryo-immobilization of these organisms quite easy. The chapter details step-by-step cryo-immobilization of plant and fungal bulk specimens by means of high pressure freezing. The following aspects are discussed in detail: (1) sampling for high pressure freezing; (2) specimen mounting and space fillers; (3) high-pressure freezing, specimen storage, and transport; and (4) freeze-substitution and embedding with epoxy or (meth) acrylate resins for morphology, immunolabeling, microanalysis, and autoradiography. In addition, it also discusses other cryo-based EM-preparation techniques. It is essential to take meticulous care with the pre- and post-freezing manipulations and to aim at a thorough understanding of the whole chain of procedures used for preparation and analysis.
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