Simultaneous detection of FISH signals and bromo-deoxyuridine incorporation in fixed tissue cultured cells

Abstract

FISH (Fluorescence in situ hybridization) is a powerful technique that detects and localises specific DNA sequences on metaphase chromosomes, interphase nuclei or chromatin fibres. When coupled to BrdU (5-Bromo 2-deoxy-uridine) labeling of newly replicated DNA, the replication properties of different DNA sequences can be analysed. However, the technique for the detection of BrdU incorporation is time consuming, and relies on acidic pH buffer treatments, that prevent use of pH sensitive fluorochromes such as FITC (Fluoro-isothiocianate) during FISH. In this work, we describe a simplified protocol that allows the simultaneous detection of FISH signals and BrdU incorporation. Since the technique does not involve paraformaldehyde for cell fixation, or formamide for denaturation of the target DNA and in post-hybridisation washes, it represents a safer alternative to classical FISH techniques. © 2009 Moralli et al.