204
Preliminary
notes
find a place as a general technique in labelchase experiments in cultured cells. We should like to thank the Cancer Research Campaign for a grant which permitted this investigation to be carried out.
References I. Schlotissek, C. Biochim biophys acta 145 (1967)
238. 2. - Eur j biochem 24 (1971) 358. 3. Murphy, W & Attardi, G, Proc natl acad sci US 70 (1973) 115. 4. Soeiro, R & Amos, H, Biochim biophys acta 129 (1966) 406. _5 HoleEkova, E. BaudySova, M & Cinnerova, 0, Exp cell res 40 (1965) 396. 6. Tobey, R A, Anderson, E C & Petersen, D F, J cell physiol70 (1967) 63. 7. Lindahl, P E & Sorenby, L, Exp cell res 43 (1966) 424. 8. Senskv, T E, Med hypotheses I (197.0 165. 9. Sensky, T E, Haines; M E & Rees, K R, Biochim bioohvs acta 407 (1975) 430. 10. Garvican, L, Cajone, F & Rees, K R. Chem-biol interactions 7 (1973) 39. 11. Penman, S, Fundamental techniques in virology (ed K Habel & N P Salzman) p. 35. Academic Press, New York (1969). 12. Plagemann, P G W, J cell physiol 77 (1971) 213. 13. -~cellbiol52fl972) 131: 14. Senskv. T E, Ph.D. Thesis. Universitv of London (1975): 15. Wiegers, U, Kramer, G, Klapproth, G, Rehpenning, W & Hilz, H, Eur j biochem 50 (1975) 557. Received November 27, 1975 Accepted December 5. 1975
Enhanced Hoechst 33258 fluorescence in plants W. G. FILION, P. MacPHERSON, D. BLAKEY, S. YEN and A. CULPEPER, Department of Botany, Erindale College, Ont. Canada
University
of Toronto,
Mississauga,
The chromosome banding produced by the benzimidazol derivative ‘Hoechst 33258’ increased in fluorescent intensity after heating, or a heating and rapid cooling procedure. In Scilla the enhanced fluorescence in both the chromocentres and chromosome bands was dramatic, whereas the response in Tulipa was not as intense.
Summary.
The introduction of the Fluorochrome ‘Hoechst 332.58’(33258 H) has provided the cytogeneticist with a stain which binds E.rpr/
Cell
Rcs YY 11976)
specifically to constitutive heterochromatin and/or repetitive DNA [l, 21; may be base specific [3,4] and characterize late replicating DNA [5]. Reports using mammalian tissue indicate that the 33258 H dye is pHdependent [6] and that differentiation may be enhanced by increased age of the slide [7]. Fluorescence with 33258 H has been reported in plants [8, 93 including Scilla sibirica [lo]; however, in contrast to animal tissue, plant tissue often requires some pretreatment prior to staining. Although 33258 H provided some linear differentiation in Scilla and Tulipa chromosomes, several modifications were conducted in order to increase the contrast and delineation of the banded regions. Since all the pre-staining modifications proved fruitless, various post-staining alternatives were performed. Materials
and Methods
Root tips were collected from Scilla sibirica and Tulipa sp., placed in 0.05% colchicine for 4 h and fixed-in acetic/ethanol 1 : 3. One-day-old slides were then subjected to the BS (Barium hydroxide/Saline) technique fill and stained in 33258 H (40 &ml) in McIlvaine’s buffer, pH 4.1 for IO sec. After-staining, the slides were rinsed and mounted in the same McIlvaine’s buffer and the cover glass sealed. Subsequently, several alternatives were applied including: (i) irradiateheat-irradiate; (ii) irradiate-heat-slow cool - irradiate; and (iii) irradiate-heat-rapid cool - irradiate. The initial irradiation was used to locate and observe a cell before treatment; elimination of this initial step did not alter the results. All irradiation occurred on the stage of a Zeiss fluorescence microscope using an HB200 lamp, a BG 12 exciter filter and observations made using a 53 barrier filter. Heat was applied to the mounted, sealed slides for about 6 set on a hot plate (120°C) or until the solution under the cover glass boiled. Rapid cooling was attained bv immediatelv placing the slide, cover glass down,. between two pieces of dry ice. In slow cooling, the slide returned to room temperature in a slide rack. Photomicrographs were taken on Kodak Tri-X film.
Results and Discussion
The 33258 H bands observed prior to the heat treatment were similar in position to those observed with Giemsa and other stains in Tulipa [l l] and Scilla [ 10, 121. Attempts to augment the linear differentiation
Prelirninury n0te.v
Fig. 1. Somatic sihiriw stained
metaphase chromosomes of Scilla with ‘Hoechst 33258’ showing the
by adjusting the pH of the 33258 H stain buffer [6] or aging the slides [7] were not successful. The post-staining treatments produced discriminate responses in Tulipa and Scilla, the latter providing a more enhanced fluorescence within the banded regions of the chromosomes. This enhanced fluorescence is extremely stable; no reduction in fluorescent intensity was observed with prolonged irradiation or storage. Application of the heat and cooling procedures prior to staining did not result in any enhanced fluorescence, indicating that enhancement occurred only in the presence of the stain. All types of 33258 H bands, i.e., centromerit, intercalary and terminal were observed in Scilfa. After heating and immediately placing over the UV light, differentiation in both the chromocentres and chromosome bands improved within seconds. This improvement was recorded as an increase limited to the field of view, that
same chromosomes dry ice enhancement
(u) before. treatment.
(h)
after
the
205
hea-
is, cells external to the circumference of the irradiated area did not improve; subsequent additional heat treatments failed to intensify the fluorescence in either area. If the slide was placed on dry ice immediately following heating, a wholesale increase in fluorescence of the banded regions and chromocentres was observed (fig. 1). When the slides were allowed to cool slowly to room temperature after heating but before irradiation. no improvement was recorded. No centromeric bands were observed in Tulipa with 33258 H as was the case with Giemsa banding [I 11. All three heat treatments exhibited some improvement in intensity in both chromocentres and bands (terminal and intercalary). The most notable change in intensity was produced by the heat-dry ice treatment whereas the slow cooling procedure provided the least. A clear difference in response to the heat treatments was observed between the two
206
Preliminary notes
species with Tulipa exhibiting little im- to DNA strandedness appears rather reprovement compared to the marked in- mote; however, if they are not, an explanacrease in intensity noted in Scilla. Species tion for the enhancement encompassing specific 33258 H staining has been reported DNA strandedness cannot be discounted in animals [l, 41; whether or not the differ- but would imply some variance between ence in fluorescent enhancement observed in vitro and in situ fluorescence patterns. here can be similarly explained is open to This work was supported by an operating grant (A6800) from the National Research Council of Canaquestion. The authors thank Mr Larocque, Canadian The most enhanced fluorescence was ob- da. Hoechst Ltd for the gift of Hoechst 33258. tained with the heat-dry ice treatment. This rapid change in temperature supposed- References ly maintains most DNA denatured by the 1. Hilwig, I & Gropp, A, Exp cell res 75 (1972) 122. heat treatment in a single-stranded or de- 2. Seth, P K & Gropp, I, Genetica 44 (1973) 485. B & Haenssler, E, Chromosoma 46 natured state. This, in addition to the pos- 3. Weisblum, (1974) 255. 4. Kucherlapati, R S, Hilwig, I, Gropp, A & Ruddle, sibility that much of the DNA already F H, Humangenetik 27 (1975) 9. existed in a single-stranded state as a result 5. Latt. S A, Somatic cell genet 1 (1975) 293. of the alkali (BS) pretreatment [13] sug- 6. Hilwig, I & Gropp, A, Exp cell res 91 (1975) 457. 7. Jalal, S M, Markvong, A & Hsu, T C, Exp cell gests that a great deal of the DNA in the res 90 (1975) 443. 8. Vosa, C G, ‘Heredity 33 (1974) 403. singlechromosomes was probably 9. Sarma, N P & Nataraian. “, A T. Hereditas 74 (1973) stranded. Recent observations have indi233. cated that 33258 H has an affinity for IO. Vosa, C G, Chromosoma 43 (1973) 269. 11. Filion, W G, Chromosoma 49 (1974) 51. double-stranded DNA [14] and that single- 12. Vosa. C G. Exe cell res 79 (1973) 463. stranded DNA, in vitro, enhances 33258 H 13. Kurn~t, D M, dytogenet celi genet 13(1974) 313. 14. Raposa, T & Natarajan, A T, Humangenetik 21 fluorescence substantially less than native (1974) 221. or reassociated DNA [15]. Therefore, if 15. Simola, K, Selander, R K, de la Chapelle, A, Corneo. G & Ginelli. E. Chromosoma 51 (1975) these characteristics [14, IS] of 33258 H 199. fluorescence are relevant to our observaReceived December 22, 1975 tions, then the possibility that the fluores- Accepted December 23, 1975 cent enhancement recorded here is related
Exptl Cell Res 99 (1976)
Preliminary
notes
find a place as a general technique in labelchase experiments in cultured cells. We should like to thank the Cancer Research Campaign for a grant which permitted this investigation to be carried out.
References I. Schlotissek, C. Biochim biophys acta 145 (1967)
238. 2. - Eur j biochem 24 (1971) 358. 3. Murphy, W & Attardi, G, Proc natl acad sci US 70 (1973) 115. 4. Soeiro, R & Amos, H, Biochim biophys acta 129 (1966) 406. _5 HoleEkova, E. BaudySova, M & Cinnerova, 0, Exp cell res 40 (1965) 396. 6. Tobey, R A, Anderson, E C & Petersen, D F, J cell physiol70 (1967) 63. 7. Lindahl, P E & Sorenby, L, Exp cell res 43 (1966) 424. 8. Senskv, T E, Med hypotheses I (197.0 165. 9. Sensky, T E, Haines; M E & Rees, K R, Biochim bioohvs acta 407 (1975) 430. 10. Garvican, L, Cajone, F & Rees, K R. Chem-biol interactions 7 (1973) 39. 11. Penman, S, Fundamental techniques in virology (ed K Habel & N P Salzman) p. 35. Academic Press, New York (1969). 12. Plagemann, P G W, J cell physiol 77 (1971) 213. 13. -~cellbiol52fl972) 131: 14. Senskv. T E, Ph.D. Thesis. Universitv of London (1975): 15. Wiegers, U, Kramer, G, Klapproth, G, Rehpenning, W & Hilz, H, Eur j biochem 50 (1975) 557. Received November 27, 1975 Accepted December 5. 1975
Enhanced Hoechst 33258 fluorescence in plants W. G. FILION, P. MacPHERSON, D. BLAKEY, S. YEN and A. CULPEPER, Department of Botany, Erindale College, Ont. Canada
University
of Toronto,
Mississauga,
The chromosome banding produced by the benzimidazol derivative ‘Hoechst 33258’ increased in fluorescent intensity after heating, or a heating and rapid cooling procedure. In Scilla the enhanced fluorescence in both the chromocentres and chromosome bands was dramatic, whereas the response in Tulipa was not as intense.
Summary.
The introduction of the Fluorochrome ‘Hoechst 332.58’(33258 H) has provided the cytogeneticist with a stain which binds E.rpr/
Cell
Rcs YY 11976)
specifically to constitutive heterochromatin and/or repetitive DNA [l, 21; may be base specific [3,4] and characterize late replicating DNA [5]. Reports using mammalian tissue indicate that the 33258 H dye is pHdependent [6] and that differentiation may be enhanced by increased age of the slide [7]. Fluorescence with 33258 H has been reported in plants [8, 93 including Scilla sibirica [lo]; however, in contrast to animal tissue, plant tissue often requires some pretreatment prior to staining. Although 33258 H provided some linear differentiation in Scilla and Tulipa chromosomes, several modifications were conducted in order to increase the contrast and delineation of the banded regions. Since all the pre-staining modifications proved fruitless, various post-staining alternatives were performed. Materials
and Methods
Root tips were collected from Scilla sibirica and Tulipa sp., placed in 0.05% colchicine for 4 h and fixed-in acetic/ethanol 1 : 3. One-day-old slides were then subjected to the BS (Barium hydroxide/Saline) technique fill and stained in 33258 H (40 &ml) in McIlvaine’s buffer, pH 4.1 for IO sec. After-staining, the slides were rinsed and mounted in the same McIlvaine’s buffer and the cover glass sealed. Subsequently, several alternatives were applied including: (i) irradiateheat-irradiate; (ii) irradiate-heat-slow cool - irradiate; and (iii) irradiate-heat-rapid cool - irradiate. The initial irradiation was used to locate and observe a cell before treatment; elimination of this initial step did not alter the results. All irradiation occurred on the stage of a Zeiss fluorescence microscope using an HB200 lamp, a BG 12 exciter filter and observations made using a 53 barrier filter. Heat was applied to the mounted, sealed slides for about 6 set on a hot plate (120°C) or until the solution under the cover glass boiled. Rapid cooling was attained bv immediatelv placing the slide, cover glass down,. between two pieces of dry ice. In slow cooling, the slide returned to room temperature in a slide rack. Photomicrographs were taken on Kodak Tri-X film.
Results and Discussion
The 33258 H bands observed prior to the heat treatment were similar in position to those observed with Giemsa and other stains in Tulipa [l l] and Scilla [ 10, 121. Attempts to augment the linear differentiation
Prelirninury n0te.v
Fig. 1. Somatic sihiriw stained
metaphase chromosomes of Scilla with ‘Hoechst 33258’ showing the
by adjusting the pH of the 33258 H stain buffer [6] or aging the slides [7] were not successful. The post-staining treatments produced discriminate responses in Tulipa and Scilla, the latter providing a more enhanced fluorescence within the banded regions of the chromosomes. This enhanced fluorescence is extremely stable; no reduction in fluorescent intensity was observed with prolonged irradiation or storage. Application of the heat and cooling procedures prior to staining did not result in any enhanced fluorescence, indicating that enhancement occurred only in the presence of the stain. All types of 33258 H bands, i.e., centromerit, intercalary and terminal were observed in Scilfa. After heating and immediately placing over the UV light, differentiation in both the chromocentres and chromosome bands improved within seconds. This improvement was recorded as an increase limited to the field of view, that
same chromosomes dry ice enhancement
(u) before. treatment.
(h)
after
the
205
hea-
is, cells external to the circumference of the irradiated area did not improve; subsequent additional heat treatments failed to intensify the fluorescence in either area. If the slide was placed on dry ice immediately following heating, a wholesale increase in fluorescence of the banded regions and chromocentres was observed (fig. 1). When the slides were allowed to cool slowly to room temperature after heating but before irradiation. no improvement was recorded. No centromeric bands were observed in Tulipa with 33258 H as was the case with Giemsa banding [I 11. All three heat treatments exhibited some improvement in intensity in both chromocentres and bands (terminal and intercalary). The most notable change in intensity was produced by the heat-dry ice treatment whereas the slow cooling procedure provided the least. A clear difference in response to the heat treatments was observed between the two
206
Preliminary notes
species with Tulipa exhibiting little im- to DNA strandedness appears rather reprovement compared to the marked in- mote; however, if they are not, an explanacrease in intensity noted in Scilla. Species tion for the enhancement encompassing specific 33258 H staining has been reported DNA strandedness cannot be discounted in animals [l, 41; whether or not the differ- but would imply some variance between ence in fluorescent enhancement observed in vitro and in situ fluorescence patterns. here can be similarly explained is open to This work was supported by an operating grant (A6800) from the National Research Council of Canaquestion. The authors thank Mr Larocque, Canadian The most enhanced fluorescence was ob- da. Hoechst Ltd for the gift of Hoechst 33258. tained with the heat-dry ice treatment. This rapid change in temperature supposed- References ly maintains most DNA denatured by the 1. Hilwig, I & Gropp, A, Exp cell res 75 (1972) 122. heat treatment in a single-stranded or de- 2. Seth, P K & Gropp, I, Genetica 44 (1973) 485. B & Haenssler, E, Chromosoma 46 natured state. This, in addition to the pos- 3. Weisblum, (1974) 255. 4. Kucherlapati, R S, Hilwig, I, Gropp, A & Ruddle, sibility that much of the DNA already F H, Humangenetik 27 (1975) 9. existed in a single-stranded state as a result 5. Latt. S A, Somatic cell genet 1 (1975) 293. of the alkali (BS) pretreatment [13] sug- 6. Hilwig, I & Gropp, A, Exp cell res 91 (1975) 457. 7. Jalal, S M, Markvong, A & Hsu, T C, Exp cell gests that a great deal of the DNA in the res 90 (1975) 443. 8. Vosa, C G, ‘Heredity 33 (1974) 403. singlechromosomes was probably 9. Sarma, N P & Nataraian. “, A T. Hereditas 74 (1973) stranded. Recent observations have indi233. cated that 33258 H has an affinity for IO. Vosa, C G, Chromosoma 43 (1973) 269. 11. Filion, W G, Chromosoma 49 (1974) 51. double-stranded DNA [14] and that single- 12. Vosa. C G. Exe cell res 79 (1973) 463. stranded DNA, in vitro, enhances 33258 H 13. Kurn~t, D M, dytogenet celi genet 13(1974) 313. 14. Raposa, T & Natarajan, A T, Humangenetik 21 fluorescence substantially less than native (1974) 221. or reassociated DNA [15]. Therefore, if 15. Simola, K, Selander, R K, de la Chapelle, A, Corneo. G & Ginelli. E. Chromosoma 51 (1975) these characteristics [14, IS] of 33258 H 199. fluorescence are relevant to our observaReceived December 22, 1975 tions, then the possibility that the fluores- Accepted December 23, 1975 cent enhancement recorded here is related
Exptl Cell Res 99 (1976)
