Reverse pattern in differential Giemsa staining of sister chromatids.

Abstract

When chromosomes are labelled for two rounds of DNA replication with BrdU and then stained with Hoechst 33258, all the sister chromatids in a metaphase spread fluoresce differentially; the unifilarly substituted (TB) chromatids fluoresce more brightly than the bifilarly substituted (BB) sister chromatids (Latt 1973) . Some technical disadvantages involved in Latt's method have been overcome by modifications in order to make permanent Giemsa-stained cytological preparations (Korenberg and Freedlender 1974;Perry and Wolff 1974;Wolff and Perry 1974;Goto et al. 1975;Sakanishi and Takayama 1977). These modified techniques give differentially Giemsa-stained chromosomes with darkly stained TB-chromatids and faintly stained BB-chromatids. Recently, we have reported the acid-extraction technique in which the use of hot acid is combined with Giemsa staining to give sister chromatid differential staining (SCD) in a pattern which is the reverse of the results obtained by the other Giemsa techniques referred to above; namely, the TB-chromatids stain faintly but the BB-chromatids stain darkly. The reverse differential staining of sister chromatids has also been reported by Scheres et al. (1977).The present paper describes two new techniques which also produce SCD in a reverse pattern similar to that obtained by the acid-extraction technique. MATERIALS AND METHODSChinese hamster strain cells taken from diaphragm and thymus were maintained in Eagle's MEM supplemented with 10% fetal calf serum. For BrdU incorporation, cells were grown in the dark in a culture medium containing 20 pM BrdU for 26 h for two round substitutions or for 40 h for three round substitutions. Colchicine (0.5 pg Jml) was added for the last 2 h and then arrested metaphase cells were collected by pipetting. The cells were treated with 0.075 M KCI, fixed in ethanol-acetic acid (3:1), and airdried on clean microslides.Preferably several-day-old slides were used. Some slides were soaked in 0.001 % trypsin at room temperature for 5 to 10 s, rinsed briefly in deionized water, and then stained in an alkaline Giemsa solution (1.5% in 0.1 M Na2HPO4, pH 9.0) for 5 to 10 min. Other slides were incubated in 0.1 M Na2HPO4 at 37°C for 2 to 4 h, and then stained in the alkaline Giemsa solution for about 10 min.