Despite their relatively small size and the fact that they are naturally non-adherent, it is possible to obtain good immunofluorescence staining of subnuclear structures in DT40. This, combined with their genetic tractability, provides a powerful combination for the study of DNA replication and repair. Here we provide a general protocol for immunofluorescence of molecules such as RAD51 and phosphorylated histone H2Ax. We also present a modification to this protocol that allows visualisation of chromatin bound PCNA and hence sites of DNA synthesis.