CHOICE OF FIXATION AND DENATURATION FOR THE TRIPLE LABELING OF INTRA-CYTOPLASMIC ANTIGEN, BROMODEOXYURIDINE AND DNA - APPLICATION TO BONE-MARROW PLASMA-CELLS

A triple staining method of intra-cytoplasmic antigen, bromodeoxyuridi ne (BrdU), and DNA for fluorescence image analysis is described. Sever al kinds of fixation and DNA denaturation methods were tested to obtai n a technique suitable for heterogeneous tissues. The model chosen was the analysis of plasma cells in bone marrow. The fluorochromes used w ere fluorescein isothiocyanate (FITC) for intra-cytoplasmic antigens ( light chain immunoglobulins), aminomethylcoumarin acetic acid (AMCA) f or BrdU, and propidium iodide (PI) for DNA. The quality of the stainin g was analysed according to: (1) cell morphology with a good preservat ion of the chromatin structure, (2) intensity of light chains and of B rdU labelling, and (3) the quality of DNA staining judged from a DNA h istogram. For most of the analysed tissues, fixation with methanol fol lowed by 0.5% paraformaldehyde and denaturation by an NaOH concentrati on adapted to the tissue gave good results. However, in our model fixa tion by methanol, followed by methanol/acetic acid and denaturation of DNA by 0.03 N NaOH was the solely satisfactory technique. A good corr elation (P<0.001) was found with the plasma cell BrdU labelling index obtained with our reference immuno-enzymatic technique. Quantification of DNA content showed a satisfactory G1 peak coefficient of variation (CV) in diploid cells and a 4C to 2C ratio equal to 2. With this tech nique, the nuclear and cytoplasmic structures of both myeloid cells an d plasma cells were well preserved, while their sensitivity to DNA den aturation was quite different.