Abstract
Abstract The preservation of S. cerevisiae ultrastructure is not a trivial task, because the cell wall serves as a diffusion barrier during all steps of preparation. In addition, a high density of proteins in the cytoplasm and in the nucleus renders the visualization of cellular details difficult. High pressure freezing in combination with freeze substitution has proven to be an advantageous technique for the preservation of the yeast mitotic spindle (1). However, the composition of the freeze substitution “cocktail” is crucial, when immunolabeling of antigens in the sample is intended. A high concentration of crosslinking agents might improve ultrastructural preservation of the sample, but will possibly destroy the antigenicity of the target proteins. In contrast, under conditions of high labeling efficiency, the ultrastructural detail may be lost. We were interested in a method, which gives a good ultrastructural preservation of the mitotic spindle and maintains the antigenicity of proteins associated with the spindle.
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