Abstract
Summary An immunocytochemical protocol has been developed to detect DNA synthesis in plant protoplasts and cells using soybean as a model system. Cells were labelled with the thymidine analog, 5-bromo-2deoxyuridine (BrdU) and fixed for antibody treatments. Formaldehyde fixation gave better results than a mixture of methanol and acetic acid. Suspension cells required an enzyme treatment to partially remove the cell wall. Fixed protoplasts and cells were extracted with Triton X-100 and reacted with a monoclonal antibody against BrdU. The BrdU labelled nuclei were visualized with either enzyme- or fluorescein-conjugated secondary antibodies. Aphidicolin reduced the number of nuclei labelled by more than 50 % at lopg-ml-', inhibited the labelling completely at 501g- ml-', confirming that the labelling was due to replicative nuclear DNA synthesis. Immunocytochemistry is simpler than autoradiography and allows simultaneous detection of DNA and other antigens including, for example, microtubules. This technique should be widely useful in studies of plant DNA replication.
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