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

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]. 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 [1,2]. 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 [3–5], the technique presented here uses a short pre-fixation step with a mixture of paraformaldehyde and sucrose followed by a short fixation/permeabilization step with a mixture of methanol and acetone at −80 °C. Method name: Fixation technique, Keywords: Immunofluorescence, Cell lines, Cytoskeleton