Intranuclear co-location of newly replicated DNA and PCNA by simultaneous immunofluorescent labelling and confocal microscopy in MCF-7 cells

Abstract

The intranuclear distribution of newly replicated DNA and of the proliferating cell nuclear antigen (PCNA) was mapped by confocal laser scanning microscopy after simultaneous immunofluorescent labelling of incorporated bromodeoxyuridine (BrdUrd) and PCNA. A mild hydrolysis with HCl followed by an enzymic digestion of DNA was used to produce single-stranded DNA required for BrdUrd immunorevelation, since this procedure preserves PCNA antigenicity. Optical sections obtained with a laser scanning microscope clearly showed a similar distribution of PCNA and BrdUrd within the nuclei, thus confirming previous observations on parallel labelled synchronized cultures. The intranuclear distribution of PCNA and BrdUrd varies concomitantly during the S phase of MCF-7 cells.