A Simple Giemsa Method for the Differential Staining of Sister Chromatids with a Note on the Presumptive Mechanism Involved

Sakanishi, Shunichi; Takayama, Susumu

doi:10.2183/pjab.53.143

Cited by 15 publications

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“…The present procedure was similar to the methods reported by Korenberg and Freedlender (1974) and Sakanishi and Takayama (1977), the use of alkaline salt solutions being common in all these procedures. The types of resulting SCD were also identical ; TB-dark and BB-light staining.…”

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“…A., Dec. 12, 1978) Recently, semiconservative segregation of DNA in eukaryotic chromosomes has elegantly been demonstrated by applying the newly developed technique of sister chromatid differential staining (Latt 1973) . This is substantiated on the fact that when cells are cultured for two successive generations in the presence of 5-bromodeoxyuridine (BrdU) and then stained with Hoechst 33258, the chromatids consisting of unifilarly BrdU-substituted (TB) DNA fluoresce more brightly than the bifilarly substituted (BB) sister chromatids.Sister chromatid differential staining (SCD) of BrdU-substituted chromosomes has also been found to occur after Giemsa staining following a variety of appropriate pretreatments (Korenberg and Freedlender 1974; Perry and Wolf 1974;Goto et al 1975;Pathak et al 1975; Sasaki 1976;Scheres et al 1977 ;Sakanishi and Takayama 1977, ; Sakanishi 1977, 1978;Burkholder 1978). To-date, however, the underlying mechanisms of SCD produced by these various procedures have remained not clear.…”

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“…Sister chromatid differential staining (SCD) of BrdU-substituted chromosomes has also been found to occur after Giemsa staining following a variety of appropriate pretreatments (Korenberg and Freedlender 1974;Perry and Wolf 1974;Goto et al 1975;Pathak et al 1975;Sasaki 1976;Scheres et al 1977 ;Sakanishi and Takayama 1977, ;Sakanishi 1977, 1978;Burkholder 1978). To-date, however, the underlying mechanisms of SCD produced by these various procedures have remained not clear.…”

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Differential Staining of Sister Chromatid in BrdU- Substituted Chromosomes after Incubation in Alkalified NaCl Solution

Takayama

Sakanishi

1978

Proceedings of the Japan Academy. Ser. B: Physical and Biologic

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(Communicated by Saj iro MAKINO, M. J. A., Dec. 12, 1978) Recently, semiconservative segregation of DNA in eukaryotic chromosomes has elegantly been demonstrated by applying the newly developed technique of sister chromatid differential staining (Latt 1973) . This is substantiated on the fact that when cells are cultured for two successive generations in the presence of 5-bromodeoxyuridine (BrdU) and then stained with Hoechst 33258, the chromatids consisting of unifilarly BrdU-substituted (TB) DNA fluoresce more brightly than the bifilarly substituted (BB) sister chromatids.Sister chromatid differential staining (SCD) of BrdU-substituted chromosomes has also been found to occur after Giemsa staining following a variety of appropriate pretreatments (Korenberg and Freedlender 1974; Perry and Wolf 1974;Goto et al. 1975;Pathak et al. 1975; Sasaki 1976;Scheres et al. 1977 ;Sakanishi and Takayama 1977, ; Sakanishi 1977, 1978;Burkholder 1978). To-date, however, the underlying mechanisms of SCD produced by these various procedures have remained not clear.In the course of investigating the mechanisms, we have found that an incubation of BrdU-substituted chromosomes in the properly alkalified NaCI solution is capable of producing SCD of the type in which TB-chromatids stain darkly while BB-ones lightly. Material and methods.Chinese hamster strain cells of lung origin maintained in Eagle's MEM supplemented with 10% fetal calf serum were grown in the dark with 20 4eM BrdU for 26 hr (two round substitutions) or for 40 hr (three round substitutions). Colchicine (0.5 pg/ml) was added to the cultures 2 hr prior to harvest. Cells were treated with 0.075 M KCI, fixed in ethanol-acetic acid (3: 1) , and airdried. Slides were kept at room temperature for at least 24 hr prior to use, and several-day-old slides were found the best for the present method. Slides aged more than three weeks were not suitable because of the poor contrast in the resulting differential staining.The alkalified NaCI solution was prepared as follows : 1-2 M NaCI was put in a Coplin jar (common soda-lime glass container)

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Differential Staining of Sister Chromatid in BrdU- Substituted Chromosomes after Incubation in Alkalified NaCl Solution

Takayama

Sakanishi

1978

Proceedings of the Japan Academy. Ser. B: Physical and Biologic

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“…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.…”

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Reverse pattern in differential Giemsa staining of sister chromatids.

Takayama

Sakanishi

1978

The Japanese journal of genetics

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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.

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