- Email: [email protected]
Silver staining of NORs in electron microscopy
Preliminary 18. Tilney, L Cl & Gibbins, J R, Protoplasma 65 (1968) 167. 19. Wheatley, D N, J anat 105 (1969) 351. 20. -Ibid 110 (1971) 367. 21. -Ibid 113 (1972) 83. 22. Wilsman, NJ & Fletcher, T F, Anat ret 190 (1978) 871. Received June 12, 1979 Revised version received July 17, 1979 Accepted July 18, 1979
Printed in Sweden Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/79/120449-04$02.00/O
Silver staining of NORs in electron microscopy C. A. BOURGEOIS, D. HERNANDEZ-VERDUN, J. HUBERT and M. BOUTEILLE, Laboratoire de Pathologie Cellulaire, Institut BiomPdical des Cordelieu, UniversitP Pierre et Marie Curie, I/ 183 INSERM and ER 189 CNRS, Paris, France Summary. We adapted the Ag-As procedure for staining the nucleolus organizer regions (NORs) to the ultrastructural level, under conditions preserving the snecificitv of the method. The fibrillar centre and the dense fibrillar component appeared to constitute the interphasic counterpart of the NOR plus the RNA transcription sites.
Nucleolus organizer regions (NORs) in chromosomes, can be selectively stained by the Ag-As method in light microscopy [l]. Goodpasture & Bloom [l] demonstrated that this technique has a specificity based on the correlation between the chromosomal sites hybridizing with ribosomal RNA (rRNA) [2] and those staining with silver [3, 41. This technique has already been applied in light microscopy to interphasic nuclei [5, 6, 71 in which active nucleoli retained the silver grains [8, 91. However, the correlation between silver deposits and individual nucleolar components has not yet been determined. It was therefore necessary to adapt the Ag-As method to the ultrastructural level, under conditions such as to preserve its selectivity. In the 29-791813
notes
449
present paper, we describe how this procedure was put into practice. Preliminary results have already been reported [lo]. Materials
and Methods
An established human cell line (TG cells) was routinely cultured in serum supplemented with Eagle’s minimal essential medium (MEM). On the day preceding the experiments, cells were seeded into 35 mm Petri dishes. Silver staining. Cells were washed in PBS and fixed at 4°C either in Camoy’s solution (1 : 3, acetic acid : ethanol) for 3-5 min or in 1.6% glutaraldehyde in Sorensen buffer, pH 7.2, for 10 min. Cells fixed with Camoy were then gradually rehydrated in ethanol, and rinsed in distilled water for 10 min. The Ag-As procedure of Goodpasture & Bloom was then applied. Cells were flooded with 50% silver nitrate solution, Petri dishes were covered and placed under a photoflood (2800°K bulb) for 10 min in such a way as to reach 45 to 55°C. The cells were twice washed with distilled water, and the same number of drops of ammoniacal silver solution and developing solution (3 % formalin) were successively applied to cover the cells until a yellow to brown coloration was observed under a light microscope (l-2 min). Cells were then thoroughly rinsed in distilled water, dehydrated, flat embedded in Epon and sectioned face-on. Sections were either contrasted in the usual way, with uranyl acetate and lead citrate or examined noncontrasted as a control. To identify the origin of silver grain accumulation, other controls were obtained by embedding cultures submitted to staining procedures which had been stopped at different stages.
Results Ag-As silver-stained cells, fixed with Carnoy or glutaraldehyde, showed concentrations of silver particules in light areas (figs 2, 4). With Carnoy fixation, the cytoplasm and nucleus were drastically altered although nucleoli were still recognizable. Fixation with glutaraldehyde allowed better preservation of the structures, although many deposits were noted in the nucleoplasm. Possible interference of the uranyl acetate and lead citrate with the silver reaction was ruled out since non-contrasted sections showed the same deposit, restricted to similar light areas (fig. 3). When cells were only treated with the 50% silver nitrate solution no deposit was observed. Only a few grains were visible when cultures were subjected Exp
Cell
Res
123 (1979)
450
Preliminary notes
Fig. 2. Nucleolus ultrastructure after conventional fixation for electron microscopy. Fibrillar centres (arrows) are surrounded by the dense tibrillar and granular components. X33 000. Fig. 2. Section of a silver-stained nucleolus after Carnoy fixation and post-staining with uranyl acetate and lead citrate. Note silver particle concentrations in light areas (arrows). x24 500.
Exp CeNRes 123(1979)
Fig. 3. Ag-As-stained nucleolus after Camoy fixation, without post-staining with uranyl acetate or lead citrate. As in figs 2 and 4, silver deposits are located in light areas. x40000. Fig. 4. Section of a silver-stained nucleus after glutaraldehyde fixation and post-staining. Silver particule concentrations are still confined to light areas, although many deposits are visible in the nucleoplasm (arrows). x 16500.
Preliminary to the complete reaction, except for the photoflood. The duration of the second step of the treatment involving the revealing solution was a critical factor. Optimal silver accumulation in electron micrographs was confined to the yellow-stained areas. Before the reaction caused this yellow staining, the density of the grains was low. When this second-step procedure was excessively prolonged, the accumulation of grains was observed to conceal the underlying structures. Comparison of silver-stained structures with nucleoli obtained under standard conditions (fig. 1) clearly identified the nucleolar fibrillar centers as the specific areas of the silver reaction (fig. 2). The number and distribution of silver accumulation areas examined in semi-serial sections contirmed this interpretation. As for the other nucleolar components, the granular one was obviously devoid of silver accumulation, whereas the dense fibrillar component seemed to be involved in the silver reaction. Discussion The significant point of this study was that the specificity of the Ag-As NOR staining method, as demonstrated on metaphasic chromosomes after Carnoy fixation, was maintained intact under our conditions, even though ultrastructural preservation suffered. The specificity of the reaction has been ascribed to particular proteins accumulated in or around the NORs [l-7], successively defined as non-histone proteins [l l] and acidic proteins whose carboxyl groups interact with silver ions [12]. In the present case, the purpose of silver staining after aldehyde fixation, a process known to localize histones [13] was not to obtain specific
notes
45 1
characterization but to insure better preservation of the structures involved in the silver staining reaction. Several authors have demonstrated the dependence of grains and therefore of protein accumulation on the degree of NOR activation [8, 9, 14, 15, 161. Consequently, the NORs stained by the Ag-AS method under our conditions can be taken to be actively involved in transcription or to be chromosomal sites which will later undergo derepression. Localization of silver staining in fibrillar centers constitutes strong evidence that the material associated with chromosome NORs persists in nucleoli during the interphase. The fibrillar centers should therefore contain the acidic proteins revealed by AgAs NOR staining. In interphasic nucleoli, several lines of evidence based on ultrastructural morphology during nucleologenesis, favour identification of the fibrillar center with the NOR [17, 18, 191. Furthermore, early nucleolar RNA synthesis was always detected around the fibrillar center wherever autoradiography was used [ 18, 20, 21, 221. It seems likely that the dense tibrillar component also reacts to silver staining, but such reactivity is still open to question, since we found this component difficult to identify after fixation and staining. On the basis of our results and those in the literature, we feel justified in concluding that, taken together, the fibrillar center and the dense fibrillar component correspond to the NOR plus the active RNA transcription sites. This work was supported by grants from INSERM (U 183) and CNRS (ER 189) and GRECO 130.023.
References 1. Goodpasture, (1975) 37.
C & Bloom, S E, Chromosoma 53 Exp Cell
Res 123 (1979)
452 2.
3.
Preliminary
notes
Hsu, T C, Spirito, S E & Pardue, M L, Chromosoma 53 (1975) 25. Henderson, A S, Warburton, D & Atwood, K C, Proc natl acad sci US 69 (1972) 3394. Pardue, M L & Hsu, T C,‘J celi biol64 (1975) 251. Martin-DeLeon, P A, Fleming, M E & Petrosky, D L, Chromosoma 67 (1978) 245. Pelliccia, F, de Canoa, A, Belloni, G. Rocchi. A & Ferraro, M, Exp cell res i 15 (1978) 439. Schwarzacher, H G, Mikelsaar, A-V & Schnedl, W, Cytogenet cell genet 20 (1978) 24. Engel, W, Zenzes, M T & Schmid, M, Hum genet 38 (1977) 57. H~srn~n, I, Gebauer, J, Bihl, L & Grimm, T, EXD cell res 114 (1978) 263. Hebandez-Verdun, D, Hubert, J, Bourgeois, C A & Bouteille. M. Comot rend acad sci 287 (1978) 1421. ‘ Howel. W M. Chromosoma 62 f 1977) 361. Olert, J, Sawatzki, G, Kling, ‘H & Gebauer, J, Histochemistry 60 (1979) 91. MacRae, E K & Meetz, G D, J cell biol45 (1970)
conventions culturing techniques, and is at least 100 times more rapid.
t%essens, G Exp cell res 100 (1976) 88 19. Mirre, C & &hi, A, J ultrastruct res 56 (1976) 186. 20. Lepoint, A & Goessens, G, Exp cell res 11J (1978) 89. 21. Mirre, C & Stahl, A, J ultrastruct res 64 (1978) 377. 22. Hemandez-Verdun. D & Bouteille, M, J ultrastmctres (1979). ln press.
A new and efficient method has been developed for obtaining a large population of mitotic cells with minimal cost and handling. Several companies have marketed new cell culturing support systems consisting of polyacrylamide, dextran matrix or plastic spheres 120-150 pm in diameter which provide excellent support for growing quantities of attachment-dependent cells in a suspension culture environment [ 1, 21. Cells growing on these beads were treated with colcemid to block cells in mitosis. During this process, mitotic cells round up, lose contact with the bead surface, and fall off into the surrounding media. A simple filtering step allows free mitotic cells to be collected. The new method not only is less expensive, easier and faster than standard culturing in flasks, but also yields a high mitotic index. We have tested polyacrylamide, dextran matrix, and plastic surface beads and conclude that for this application the acrylamide matrix beads are superior.
Received June 29, 1979 Accepted July 23, 1979
Materials
4. 5. 6. 7. 8.
9. 10. 11. 12. 13.
17c
‘,J
.
14. Miller, D A, Dev, V G, Tantravahi, R & Miller, 0 J, Exp cell res 101 (1976) 235. 15. Miller, 0 J, Miller, D A, Dev, V G, Tantravahi, R & Croce, C M, Proc natl acad sci US 73 (1976) 4531. 16. Tantravahi, R, Miller, D A, D’Ancona, G, Croce, C M & Miller, 0 J, Exp cell res 119 (1979) 387. 17. Goessens, G & Lepoint, A, Exp cell res 87 (1974) 18.
_...
Printed in Sweden Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/?9/[email protected]/0
Mitotic cell ~pul~tions micro-carrier culturing KIM J. MITCHELL
obtained from a system
and WAYNE
ment of Cell Biology, Baylor Houston, TX 77030, USA
WRAY,’ Deparr-
College
of Medicine,
Summary. Colcemid treatment of cell culture beads confluent with cells released a mitotic population of cells which was as efficient as that obtained by conventional shake-off procedures. The bead system provides an easy and efftcient means for harvesting large quantities of mitotic cells, costs about one-tenth as much as Exp Cell Res 123 (1979)
Bio-Carrier nolvacrvlamide beads (2 a beads in 100 ml HEPES buffer, Bio-Rad LaboratoGes, Richmond, Calif.). Sunerbeads Micro-Carriers (1 g dextran bead in IO& ml PBS, Flow Laboratories, Rockville, Md), Cytodex (100 g and 500 g dry dextran bead packs, Pharmacia Fine Chemicals, Uppsala), and Biosilon plastic beads (Nunc) were inoculated with cells from a Chinese hamster ovary cell line (CHO) or from a human tumor cell line (HeLa). Cell cultures were mown in McCov’s 5a medium with L-~lu~mine TKC Biological, Inc., Lenexa, KS) supplemented with 10% fetal bovine serum (KC Biological), 0.045 mg/ml gentamycin (Schering Corp., Kenilworth, N.J.), and atmospheric conditions were controlled with a 10% CO, gas mixture. The bead cultures were grown in spinner flasks (Bellco Glass, Inc., Vineland, N.J.), stirring at 65 rpm. Mitotic populations were obtained following the addition of 0.06 p&/ml colcemid (CIBA Pharmaceutical Co., Summit, N.J.) for 7 h (CHO) or 20 h (HeLa). Nylon mesh 88 pm pore size (Small r To whom reprint requests should be addressed.
Printed in Sweden Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/79/120449-04$02.00/O
Silver staining of NORs in electron microscopy C. A. BOURGEOIS, D. HERNANDEZ-VERDUN, J. HUBERT and M. BOUTEILLE, Laboratoire de Pathologie Cellulaire, Institut BiomPdical des Cordelieu, UniversitP Pierre et Marie Curie, I/ 183 INSERM and ER 189 CNRS, Paris, France Summary. We adapted the Ag-As procedure for staining the nucleolus organizer regions (NORs) to the ultrastructural level, under conditions preserving the snecificitv of the method. The fibrillar centre and the dense fibrillar component appeared to constitute the interphasic counterpart of the NOR plus the RNA transcription sites.
Nucleolus organizer regions (NORs) in chromosomes, can be selectively stained by the Ag-As method in light microscopy [l]. Goodpasture & Bloom [l] demonstrated that this technique has a specificity based on the correlation between the chromosomal sites hybridizing with ribosomal RNA (rRNA) [2] and those staining with silver [3, 41. This technique has already been applied in light microscopy to interphasic nuclei [5, 6, 71 in which active nucleoli retained the silver grains [8, 91. However, the correlation between silver deposits and individual nucleolar components has not yet been determined. It was therefore necessary to adapt the Ag-As method to the ultrastructural level, under conditions such as to preserve its selectivity. In the 29-791813
notes
449
present paper, we describe how this procedure was put into practice. Preliminary results have already been reported [lo]. Materials
and Methods
An established human cell line (TG cells) was routinely cultured in serum supplemented with Eagle’s minimal essential medium (MEM). On the day preceding the experiments, cells were seeded into 35 mm Petri dishes. Silver staining. Cells were washed in PBS and fixed at 4°C either in Camoy’s solution (1 : 3, acetic acid : ethanol) for 3-5 min or in 1.6% glutaraldehyde in Sorensen buffer, pH 7.2, for 10 min. Cells fixed with Camoy were then gradually rehydrated in ethanol, and rinsed in distilled water for 10 min. The Ag-As procedure of Goodpasture & Bloom was then applied. Cells were flooded with 50% silver nitrate solution, Petri dishes were covered and placed under a photoflood (2800°K bulb) for 10 min in such a way as to reach 45 to 55°C. The cells were twice washed with distilled water, and the same number of drops of ammoniacal silver solution and developing solution (3 % formalin) were successively applied to cover the cells until a yellow to brown coloration was observed under a light microscope (l-2 min). Cells were then thoroughly rinsed in distilled water, dehydrated, flat embedded in Epon and sectioned face-on. Sections were either contrasted in the usual way, with uranyl acetate and lead citrate or examined noncontrasted as a control. To identify the origin of silver grain accumulation, other controls were obtained by embedding cultures submitted to staining procedures which had been stopped at different stages.
Results Ag-As silver-stained cells, fixed with Carnoy or glutaraldehyde, showed concentrations of silver particules in light areas (figs 2, 4). With Carnoy fixation, the cytoplasm and nucleus were drastically altered although nucleoli were still recognizable. Fixation with glutaraldehyde allowed better preservation of the structures, although many deposits were noted in the nucleoplasm. Possible interference of the uranyl acetate and lead citrate with the silver reaction was ruled out since non-contrasted sections showed the same deposit, restricted to similar light areas (fig. 3). When cells were only treated with the 50% silver nitrate solution no deposit was observed. Only a few grains were visible when cultures were subjected Exp
Cell
Res
123 (1979)
450
Preliminary notes
Fig. 2. Nucleolus ultrastructure after conventional fixation for electron microscopy. Fibrillar centres (arrows) are surrounded by the dense tibrillar and granular components. X33 000. Fig. 2. Section of a silver-stained nucleolus after Carnoy fixation and post-staining with uranyl acetate and lead citrate. Note silver particle concentrations in light areas (arrows). x24 500.
Exp CeNRes 123(1979)
Fig. 3. Ag-As-stained nucleolus after Camoy fixation, without post-staining with uranyl acetate or lead citrate. As in figs 2 and 4, silver deposits are located in light areas. x40000. Fig. 4. Section of a silver-stained nucleus after glutaraldehyde fixation and post-staining. Silver particule concentrations are still confined to light areas, although many deposits are visible in the nucleoplasm (arrows). x 16500.
Preliminary to the complete reaction, except for the photoflood. The duration of the second step of the treatment involving the revealing solution was a critical factor. Optimal silver accumulation in electron micrographs was confined to the yellow-stained areas. Before the reaction caused this yellow staining, the density of the grains was low. When this second-step procedure was excessively prolonged, the accumulation of grains was observed to conceal the underlying structures. Comparison of silver-stained structures with nucleoli obtained under standard conditions (fig. 1) clearly identified the nucleolar fibrillar centers as the specific areas of the silver reaction (fig. 2). The number and distribution of silver accumulation areas examined in semi-serial sections contirmed this interpretation. As for the other nucleolar components, the granular one was obviously devoid of silver accumulation, whereas the dense fibrillar component seemed to be involved in the silver reaction. Discussion The significant point of this study was that the specificity of the Ag-As NOR staining method, as demonstrated on metaphasic chromosomes after Carnoy fixation, was maintained intact under our conditions, even though ultrastructural preservation suffered. The specificity of the reaction has been ascribed to particular proteins accumulated in or around the NORs [l-7], successively defined as non-histone proteins [l l] and acidic proteins whose carboxyl groups interact with silver ions [12]. In the present case, the purpose of silver staining after aldehyde fixation, a process known to localize histones [13] was not to obtain specific
notes
45 1
characterization but to insure better preservation of the structures involved in the silver staining reaction. Several authors have demonstrated the dependence of grains and therefore of protein accumulation on the degree of NOR activation [8, 9, 14, 15, 161. Consequently, the NORs stained by the Ag-AS method under our conditions can be taken to be actively involved in transcription or to be chromosomal sites which will later undergo derepression. Localization of silver staining in fibrillar centers constitutes strong evidence that the material associated with chromosome NORs persists in nucleoli during the interphase. The fibrillar centers should therefore contain the acidic proteins revealed by AgAs NOR staining. In interphasic nucleoli, several lines of evidence based on ultrastructural morphology during nucleologenesis, favour identification of the fibrillar center with the NOR [17, 18, 191. Furthermore, early nucleolar RNA synthesis was always detected around the fibrillar center wherever autoradiography was used [ 18, 20, 21, 221. It seems likely that the dense tibrillar component also reacts to silver staining, but such reactivity is still open to question, since we found this component difficult to identify after fixation and staining. On the basis of our results and those in the literature, we feel justified in concluding that, taken together, the fibrillar center and the dense fibrillar component correspond to the NOR plus the active RNA transcription sites. This work was supported by grants from INSERM (U 183) and CNRS (ER 189) and GRECO 130.023.
References 1. Goodpasture, (1975) 37.
C & Bloom, S E, Chromosoma 53 Exp Cell
Res 123 (1979)
452 2.
3.
Preliminary
notes
Hsu, T C, Spirito, S E & Pardue, M L, Chromosoma 53 (1975) 25. Henderson, A S, Warburton, D & Atwood, K C, Proc natl acad sci US 69 (1972) 3394. Pardue, M L & Hsu, T C,‘J celi biol64 (1975) 251. Martin-DeLeon, P A, Fleming, M E & Petrosky, D L, Chromosoma 67 (1978) 245. Pelliccia, F, de Canoa, A, Belloni, G. Rocchi. A & Ferraro, M, Exp cell res i 15 (1978) 439. Schwarzacher, H G, Mikelsaar, A-V & Schnedl, W, Cytogenet cell genet 20 (1978) 24. Engel, W, Zenzes, M T & Schmid, M, Hum genet 38 (1977) 57. H~srn~n, I, Gebauer, J, Bihl, L & Grimm, T, EXD cell res 114 (1978) 263. Hebandez-Verdun, D, Hubert, J, Bourgeois, C A & Bouteille. M. Comot rend acad sci 287 (1978) 1421. ‘ Howel. W M. Chromosoma 62 f 1977) 361. Olert, J, Sawatzki, G, Kling, ‘H & Gebauer, J, Histochemistry 60 (1979) 91. MacRae, E K & Meetz, G D, J cell biol45 (1970)
conventions culturing techniques, and is at least 100 times more rapid.
t%essens, G Exp cell res 100 (1976) 88 19. Mirre, C & &hi, A, J ultrastruct res 56 (1976) 186. 20. Lepoint, A & Goessens, G, Exp cell res 11J (1978) 89. 21. Mirre, C & Stahl, A, J ultrastruct res 64 (1978) 377. 22. Hemandez-Verdun. D & Bouteille, M, J ultrastmctres (1979). ln press.
A new and efficient method has been developed for obtaining a large population of mitotic cells with minimal cost and handling. Several companies have marketed new cell culturing support systems consisting of polyacrylamide, dextran matrix or plastic spheres 120-150 pm in diameter which provide excellent support for growing quantities of attachment-dependent cells in a suspension culture environment [ 1, 21. Cells growing on these beads were treated with colcemid to block cells in mitosis. During this process, mitotic cells round up, lose contact with the bead surface, and fall off into the surrounding media. A simple filtering step allows free mitotic cells to be collected. The new method not only is less expensive, easier and faster than standard culturing in flasks, but also yields a high mitotic index. We have tested polyacrylamide, dextran matrix, and plastic surface beads and conclude that for this application the acrylamide matrix beads are superior.
Received June 29, 1979 Accepted July 23, 1979
Materials
4. 5. 6. 7. 8.
9. 10. 11. 12. 13.
17c
‘,J
.
14. Miller, D A, Dev, V G, Tantravahi, R & Miller, 0 J, Exp cell res 101 (1976) 235. 15. Miller, 0 J, Miller, D A, Dev, V G, Tantravahi, R & Croce, C M, Proc natl acad sci US 73 (1976) 4531. 16. Tantravahi, R, Miller, D A, D’Ancona, G, Croce, C M & Miller, 0 J, Exp cell res 119 (1979) 387. 17. Goessens, G & Lepoint, A, Exp cell res 87 (1974) 18.
_...
Printed in Sweden Copyright @ 1979 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/?9/[email protected]/0
Mitotic cell ~pul~tions micro-carrier culturing KIM J. MITCHELL
obtained from a system
and WAYNE
ment of Cell Biology, Baylor Houston, TX 77030, USA
WRAY,’ Deparr-
College
of Medicine,
Summary. Colcemid treatment of cell culture beads confluent with cells released a mitotic population of cells which was as efficient as that obtained by conventional shake-off procedures. The bead system provides an easy and efftcient means for harvesting large quantities of mitotic cells, costs about one-tenth as much as Exp Cell Res 123 (1979)
Bio-Carrier nolvacrvlamide beads (2 a beads in 100 ml HEPES buffer, Bio-Rad LaboratoGes, Richmond, Calif.). Sunerbeads Micro-Carriers (1 g dextran bead in IO& ml PBS, Flow Laboratories, Rockville, Md), Cytodex (100 g and 500 g dry dextran bead packs, Pharmacia Fine Chemicals, Uppsala), and Biosilon plastic beads (Nunc) were inoculated with cells from a Chinese hamster ovary cell line (CHO) or from a human tumor cell line (HeLa). Cell cultures were mown in McCov’s 5a medium with L-~lu~mine TKC Biological, Inc., Lenexa, KS) supplemented with 10% fetal bovine serum (KC Biological), 0.045 mg/ml gentamycin (Schering Corp., Kenilworth, N.J.), and atmospheric conditions were controlled with a 10% CO, gas mixture. The bead cultures were grown in spinner flasks (Bellco Glass, Inc., Vineland, N.J.), stirring at 65 rpm. Mitotic populations were obtained following the addition of 0.06 p&/ml colcemid (CIBA Pharmaceutical Co., Summit, N.J.) for 7 h (CHO) or 20 h (HeLa). Nylon mesh 88 pm pore size (Small r To whom reprint requests should be addressed.