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

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

doi:10.1007/s00418-021-02070-0

Martin Schauflinger, Christine Kranz + Show 3 more

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https://doi.org/10.1007/s00418-021-02070-0

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Abstract

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.

Highlights

Rapid cryofixation by high-pressure freezing (HPF) followed by freeze-substitution (FS) results in superior preservation of biological samples compared with conventional electron microscopy (EM) sample preparation protocols that rely on chemical fixation and dehydration of cellular structures at ambient temperatures (Dahl and Staehelin 1989; Shiurba 2001; Zechmann et al 2007)
We show that KMnO4 is an excellent en bloc stain for freeze-substituted samples, and provides high inherent electron density to cellular ultrastructure when used in combination with uranyl acetate
We show that KMnO4 in combination with uranyl acetate in the FS solution can effectively replace OsO4 as a fixative and staining agent in different cultured cells

Summary

Introduction

Rapid cryofixation by high-pressure freezing (HPF) followed by freeze-substitution (FS) results in superior preservation of biological samples compared with conventional electron microscopy (EM) sample preparation protocols that rely on chemical fixation and dehydration of cellular structures at ambient temperatures (Dahl and Staehelin 1989; Shiurba 2001; Zechmann et al 2007). One of the most important and demanding tasks of biological EM is the visualization of cellular membranes. Depending on the specific biological system and methodology used, it can be challenging to achieve good membrane staining, especially in high-pressure frozen cells (Walther and Ziegler 2002). It is necessary to treat sectioned biological specimens with heavy metal salts to achieve sufficient contrast (Reynolds 1963; Ellis 2014).

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