Choice of fixation and denaturation for the triple labelling of intra-cytoplasmic antigen,bromodeoxyuridine and DNA

A triple staining method of intra-cytoplasmic antigen, bromodeoxyuridine (BrdU), and DNA for fluorescence image analysis is described. Several kinds of fixation and DNA denaturation methods were tested to obtain a technique suitable for heterogeneous tissues. The model chosen was the analysis of plasma cells in bone marrow. The fluorochromes used were fluorescein isothiocyanate (FITC) for intra-cytoplasmic antigens (light chain immunoglobulins), aminomethylcoumarin acetic acid (AMCA) for BrdU, and propidium iodide (PI) for DNA. The quality of the staining was analysed according to: (1) cell morphology with a good preservation of the chromatin structure, (2) intensity of light chains and of BrdU labelling, and (3) the quality of DNA staining judged from a DNA histogram. For most of the analysed tissues, fixation with methanol followed by 0.5% paraformaldehyde and denaturation by an NaOH concentration adapted to the tissue gave good results. However, in our model fixation by methanol, followed by methanol/acetic acid and denaturation of DNA by 0.03 N NaOH was the solely satisfactory technique. A good correlation (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 technique, the nuclear and cytoplasmic structures of both myeloid cells and plasma cells were well preserved, while their sensitivity to DNA denaturation was quite different.