High-throughput screening of mitotic mammalian cells for electron microscopy using classic histological dyes

Abstract

We introduce a new workflow that allows screening and selection of staged mammalian cells in mitosis prior to subsequent electron microscopy. We mainly describe four improved steps of specimen preparation. Firstly, we describe a method to efficiently enrich mammalian cells and attach them to sapphire discs; secondly, we report on the use of 3D-printed containers to seed cells on coated sapphire discs for high-pressure freezing; thirdly, we take advantage of a specimen carrier that allows for an upside-down placing of sapphire discs without a second carrier or spacer ring to close the "sandwich"; and fourthly, we use histological dyes to stain DNA/chromatin during freeze-substitution. Out of 14 tested histological dyes, we routinely use four of them for visual inspection of mitotic cells by light microscopy. Applying this streamlined workflow, HeLa cells at different stages of mitosis can be selected for further ultrastructural analysis. The practical aspects of this approach will be discussed herein.