Double and triple staining methods for studying the proliferative activity of human B and T lymphoid cells

New methods for double and triple colour labelling using monoclonal antibodies to the proliferation-associated markers 5-bromo-2'-deoxyuridine (BrdU) and Ki67 are described. In order to make incorporated BrdU accessible to most anti-BrdU antibodies, mild denaturation of the DNA is needed, and this is usually obtained by exposing the cells to acid or base. This procedure destroys most cellular antigens, including nuclear TdT and Ki67. In this study we show that fixation in cold methanol, instead of 70% ethanol, for 30 min followed by immersion in 7 X 10(-3) N NaOH for 10-15 s allows BrdU staining with the simultaneous detection of nuclear, cytoplasmic and membrane antigens as well as preservation of morphological detail. This method is optimal for detection of nuclear Ki67 and TdT. These reagents together with antibodies to membrane antigens can be included in triple colour labelling using second layers conjugated to FITC, TRITC and colloidal gold. With these methods it is now possible to characterize the phenotype of dividing cell populations such as precursors in central lymphoid tissues and germinal centre blasts in peripheral lymphoid organs.