Immunohistochemical Detection of Bromodeoxyuridine-Labeled Nuclei for In Vivo Cell Kinetic Studies

The classical technique for identifying cells engaged in DNA synthesis is by their uptake of [³ H]-thymidine, detected using autoradiography. However, this method can be inconvenient, as specialized darkroom and radioisotope facilities are required, with the potential health hazard that handling isotopes entails. Bromodeoxyundine (BrdU), the halogenated 5-substituted derivative of deoxyuridine, is a thymidine analog specifically incorporated into the DNA of proliferating cells during S phase. This is now a well-established alternative to [³ H] thymidine, since it has been shown that labeling indices for the two molecules are the same (1 ,2 ) The development of a monoclonal antibody (3 ) that recognizes BrdU incorporated into single-stranded DNA has resulted in several techniques using immunocytochemical staining to detect incorporated BrdU in frozen, paraffin- and plastic-embedded sections of tissue by light microscopy. It has also proved extremely valuable for studies in conjunction with flow cytometry and even, for in vivo studies of human tumor cell kinetics (see Chapter 26). We describe here a method to detect DNA synthesis by in vivo labeling of nuclei with BrdU, followed by indirect immunological detection in paraffin-embedded tissue (4 )