Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution.

Martin Schauflinger, Tim Bergner, Gregor Neusser, Christine Kranz, Clarissa Read

Histochemistry and cell biology 2022 Apr

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High-pressure freezing followed by freeze-substitution is a valuable method for ultrastructural analyses of resin-embedded biological samples. The visualization of lipid membranes is one of the most critical aspects of any ultrastructural study and can be especially challenging in high-pressure frozen specimens. Historically, osmium tetroxide has been the preferred fixative and staining agent for lipid-containing structures in freeze-substitution solutions. However, osmium tetroxide is not only a rare and expensive material, but also volatile and toxic. Here, we introduce the use of a combination of potassium permanganate, uranyl acetate, and water in acetone as complementing reagents during the freeze-substitution process. This mix imparts an intense en bloc stain to cellular ultrastructure and membranes, which makes poststaining superfluous and is well suited for block-face imaging. Thus, potassium permanganate can effectively replace osmium tetroxide in the freeze-substitution solution without sacrificing the quality of ultrastructural preservation. © 2022. The Author(s).

Citation

Martin Schauflinger, Tim Bergner, Gregor Neusser, Christine Kranz, Clarissa Read. Potassium permanganate is an excellent alternative to osmium tetroxide in freeze-substitution. Histochemistry and cell biology. 2022 Apr;157(4):481-489