Mechanisms involved in differential staining of BrdU substituted chromatids or chromosome regions

PHA stimulated peripheral blood lymphocytes were in vitro labelled with BrdU for 72 h. before cells gathering. Chromosome spreads were obtained by a standard method and some different techniques were used for differential staining of sister chromatids: a) denaturation of BrdU substituted DNA by thermal (90 degrees C) and alkaline (pH 9) treatments: b) UV-induced photolysis of substituted DNA pretreated with intercalating fluorescent agents (acridinorange or 33258 Hoechst) followed by heat treatment (60 degrees C); c) UV-induced photolysis and heat treatment (60 degrees C) without any fluorescent dyes or buffers. With this technique, incorporation banding was also obtained in cells labelled with high concentrations of BrdU during the last round of DNA replication. In all these methods the slides were finally coloured with Giemsa solution. The results obtained with each of the techniques used were compared and the possible molecular mechanisms involved were discussed. Even if many experimental factors could be of importance for revealing the BrdU substituted chromatids or chromosome segments, UV-irradiation and/or heat treatment appear to be mandatory for both differential staining of sister chromatids and for incorporation banding.