In vitro immunohistochemical localization of S-phase cells by a monoclonal antibody to bromodeoxyuridine

Thin viable slices of normal or pathological human tissues were incubated in vitro with bromodeoxyuridine (BrdU). Later, cryostatic sections and histological sections from the same samples embedded in paraffin were examined by an immunohistochemical method using a monoclonal antibody anti-bromodeoxyuridine (anti-BrdU-MAb): on both cryostatic and histological sections, the nuclei of the S-phase cells proved positive. The optimization of the technique depends on the concentration of bromodeoxyuridine in the culture medium (160 microM), the duration of incubation (not less than two h), the method of DNA denaturation (2N or 4N HCl) and the dilution of the anti-BrdU-MAb (1:50). In vitro, immunohistochemical application of the BrdU/anti-BrdU-MAb method permits a quantitative assessment of the proliferative activity of a tissue as well as the direct location of the actively replicating cells in histological sections.