Immunological staining of BrdUrd : evaluation of DNA denaturation using DNase

In cell kinetic research, there is an increasing interest in the use of immunohistochmistry for the detection of bromo-deoxyuridine (BrdUrd), which S-phase cells have incorporated into their DNA. In order to make the incorporated BrdUrd accessible to an anti-BrdUrd antibody (B-44), DNA denaturation is required. Usually this has been accomlished by exposing the cells to acid. However, the procedure using acid may destory cellular membrane antigens as well as morphological details. BU-1 antibody, when used in combination with DNase for DNA denaturation, reacts with the incorporated BrdUrd without using acid. In this study, the efficacy of BU-1 antibody was investigated and compared with that of B-44 antibody. Ten tumor speciments were used for this study. Under varying conditions (one or three atomospheric pressure during the BrdUrd lebeling and different temperture during the incubation of BU-1 antibody), the tumor tissue was labeled with BrdUrd in vitro and the each sections were immunohistochemically stained with BU-1 antibody or B-44 antibody. Three atmosppheric pressure was required for labeling cells with BrdUrd in vitro. The stainability of BrdUrd was enhanced, when tissue specimens were incubated at 37℃. The BU-1 antibody provided results equivalent to the B-44 antibody. Double staining of BrdUrd and epithelial membrane antigen (EMA) was carried out. EMA and cellular morphology were better preserved in preparations stained with BU-1 antibody than with B-44 antibody.