A NEW IMMUNOCYTOCHEMICAL TECHNIQUE FOR ULTRASTRUCTURAL ANALYSIS OF DNA-REPLICATION IN PROLIFERATING CELLS AFTER APPLICATION OF 2 HALOGENATED DEOXYURIDINES

We describe a colloidal gold immunolabeling technique for electron mic roscopy which allows one to differentially visualize portions of DNA r eplicated during different periods of S-phase. This was performed by i ncorporating two halogenated deoxyuridines (IdUrd and CldUrd) into Chi nese hamster cells and, after cell processing, by detecting them with selected antibodies. This technique, using in particular appropriate b locking solutions and also Tris buffer with a high salt concentration and 1% Tween-20, prevents nonspecific background and crossreaction of both antibodies. Controls such as digestion with DNase and specific st aining of DNA with osmium ammine show that labeling corresponds well t o replicated DNA. Different patterns of labeling distribution, reflect ing different periods of DNA replication during S-phase, were characte rized. Cells in early S-phase display a diffuse pattern of labeling wi th many spots, whereas cells in late S-phase show labeling confined to larger domains, often at the periphery of the nucleus or associated w ith the nucleolus. The good correlation between our observations and p revious double labeling results in immunofluorescence also proved the technique to be reliable.