A simple and fast method for fixation of cultured cell lines that preserves cellular structures containing gamma-tubulin

MethodsX
Maria Alvarado-Kristensson

Abstract

When using fluorescence microscope techniques to study cells, it is essential that the cell structure and contents are preserved after preparation of the samples, and that the preparation method employed does not create artefacts that can be perceived as cellular structure/components. γ-Tubulin forms filaments that in some cases are immunostained with an anti-γ-tubulin antibody, but this immunostaining is not reproducible [[1], [2]]. In addition, the C terminal region of γ-tubulin (green fluorescence protein tagged [GFP]-γ-tubulin334--449) forms cytosolic GFP-labeled structures, which can easily be imaged in live cells but are not preserved in fixed cells [[1], [3]]. The purpose of this study was to identify a fixation technique that preserves cellular constituents containing γ-tubulin. •This protocol describes a method that preserves γ-tubulin-containing structures in fixed cells.•The technique entails two-step fixation. A pre-fixation step using paraformaldehyde is followed by a final fixation and permeabilization step performed at -80 °C.•In comparison with other methodology for fixation [[4], [5], [6]], the technique presented here uses a short pre-fixation step with a mixture of paraformaldehyde and sucrose followed by a s...Continue Reading

Citations

Oct 24, 2018·International Journal of Molecular Sciences·Catalina Ana RossellóMaria Alvarado Kristensson
Jan 29, 2020·Heliyon·Maria Alvarado-Kristensson
Oct 30, 2020·Cancers·Matthieu Corvaisier, Maria Alvarado-Kristensson
Apr 4, 2021·Cells·Maria Alvarado Kristensson
Jun 24, 2021·Communications Biology·Matthieu CorvaisierMaria Alvarado-Kristensson

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