The challenges of sample preparation can limit a researcher's selection of
transmission electron microcopy (TEM) for analysis of yeast. However, with
the exception of thin sectioning, preparation of well-fixed and infiltrated
samples of yeast cells is achievable by any reasonably equipped laboratory
. This review presents a general overview of TEM sample preparation methods
and detailed protocols for chemical fixation of yeast for ultrastructural
analysis and immunolabeling. For ultrastructural analysis, the most commonl
y used chemical fixation involves treatment with glutaraldehyde followed by
either potassium permanganate or osmium. Prior to osmium postfixation, the
cell wall must be enzymatically digested to allow optimal fixation and emb
edding. Freeze substitution methods continue to provide the highest quality
of fixation, but equipment needed for these protocols is not generally ava
ilable to many labs. The low viscosity of Spurr's resin makes it the resin
of choice for ultrastructure studies. Immunoelectron microscopy has enjoyed
great success in analysis of yeast molecular organization. For immunoelect
ron microscopy, glutaraldehyde/formaldehyde-fixed cells are embedded in LR
White resin. The thin sections are then treated in much the same way as an
immunoblot: following blocking, they are incubated in primary antiserum, wa
shed, and then incubated in gold-labeled secondary antiserum. (C) 2000 Wile
y-Liss. Inc.