DNA synthesis in cultured hepatocytes. Hepatocytes of the indicated genotypes were cultured in the presence and absence of EGF as indicated and labeled with 5 μCi tritiated thymidine or 10 μM BrdU for 24 hours before harvest on different days in culture. (a) Tritiated thymidine incorporation. The data presented are means ± SEM and are representative of five separate experiments with similar results. (b) BrdU incorporation. For each sample, 300–400 nuclei were scored for BrdU incorporation. (c) Immunofluorescence staining for BrdU incorporation in hepatocytes. Nuclei were stained with Hoechst 33258, and BrdU was detected with anti-BrdU primary and FITC-labeled secondary Ab’s. Cells were photographed at ×400 with an Olympus IX70 inverted fluorescence microscope (Olympus America Inc., Melville, New York, USA) and digital camera (cooled CCD camera Photometrics PXL; Roper Instruments, Tuscon, Arizona, USA). All data presented are from the same animals.