Electron microscopy and high-pressure freezing of Arabidopsis.

In this chapter, we will discuss methods and protocols for high-pressure freezing (HPF) and freeze substitution (FS) to examine Arabidopsis tissues by transmission electron microscopy (TEM). By use of HPF in combination with FS, it is possible to obtain Arabidopsis samples that are far better preserved for both ultrastructural analysis and immunogold labeling than by conventional chemical fixation. Like other cryofixation methods, ice crystal growth is still a problem in HPF if samples are too thick (> 200 μm) or if their water content is too high. Furthermore, damage done to cells/tissues prior to freezing cannot be "reverted" by HPF. In general, FS of plant tissues is more difficult than that of nonplant tissues because plant cell walls impede removal of water from the enclosed cells as well as from the walls themselves. To overcome these challenges, we describe the details of a HPF, FS, and resin-embedding protocol for Arabidopsis tissues here. In addition, the generation of ribbons of serial sections from Arabidopsis TEM blocks, three-dimensional (3D) analysis of organelle shapes and distribution within the tissue, and immunogold labeling are also explained. The Arabidopsis research community has developed many research tools to investigate gene functions such as knockout mutant lines, antibodies, and transgenic lines expressing epitope-tagged proteins. The TEM techniques explained here have been combined with these tools to elucidate how a particular gene of interest functions in the Arabidopsis cell.