Immunofluorescent staining of poly(ADP-ribose) in situ in HeLa cell chromosomes in the M phase.

Randomly and synchronously growing HeLa cells were tested for poly(ADP-ribose) by direct and indirect immunofluorescent antibody techniques. Fluorescence of poly(ADP-ribose) was seen only in the nuclei of intact cells when the direct immunofluorescent antibody technique was used but in both the nuclei and cytoplasm when the indirect immunofluorescent antibody technique was used; fluorescence in the cytoplasm was nonspecific. When randomly or synchronously growing HeLa cells were fixed in acetone and treated with DNase I before incubation with fluorescein-labeled antibody, intense fluorescence was observed only in the nuclei when the direct immunofluorescent staining technique was used. Addition of 3-aminobenzamide, a potent inhibitor of poly(ADP-ribose) polymerase, with the DNase I completely abolished the fluorescence in the nuclei of synchronously and randomly growing HeLa cells, except in M-phase nuclei. These results suggest that poly(ADP-ribose) can be synthesized even in the nuclei of acetone-fixed HeLa cells from "endogenous NAD+" during incubation with fluorescent antibody and also that the fluorescence of chromosomes of HeLa cells in the M phase is, in fact, due to the in situ presence of poly(ADP-ribose), not to poly(ADP-ribose) synthesized during incubation with antibody.