Preliminary
rived from these labelling experiments is that, at the exocytosis sites, cell membrane materials flow in a centropetal direction; this mechanism allows for subsequent membrane resealing. Results are summarized in fig. 4 (A-G): Upon Ca*+-influx (B, [lo]) the apical trichocyst membrane and the cell membrane portion encircled by a “ring” undergo fragmentation (C), allowing the trichocyst contents to be discharged (0). At this stage the cell membrane and the trichocyst membrane have fused with each other within “rings” (II). Subsequently the exocytosis channels become narrower as some cell membrane and underlying cytoplasmic materials spread to within the “rings” (E). Some surface label spreads from the cell membrane onto the uppermost trichocyst membrane region (F). As the labelling density of the cell membrane around exocytosis sites appears “undiluted” at all stages, this indicates a centropetal diffusion of cell membrane materials, which then allows the cell membrane and the trichocyst membrane to become completely resealed and to become finally detached from each other (G). All this evidence leads to the conclusion that with Paramecium cells exocytosis involves retrieval of the secretory vesicle membranes rather than their integration into the cell membrane. This conclusion is in agreement with a recent morphological analysis by Hausmann & Allen [ 161. This study was supported by grant no. 2923 from the “Osterreichische Fonds zur Forderung der wissenschaftlichen Forschung”.
Rqferences 1. Winkler, H, Phil tram roy sot London B 261(1971) 293.
2. Satir, B, Schooley, C & Satir, P. J cell biol 56 (1973) 153. 3. Satir, B, Sci Am 233 (1975) 28.
4. Van Wagtendonk, W J, Paramecium. A current survey. Elsevier, Amsterdam, London, New York (19743.
mxes
435
5. Bachmann, L. Schmitt, W W Bi Plattner, H, Proc 5th Em congr electron microscopy, Manchester (ed V E Cosslett) D. 244. The Institute of Phvsics, . London, Bristol (f972). 6. Plattner. H, Miller, F & Bachmann, L. J cell sci !3 (1973) 687. 7. Plattner, H, Nature 252 (1974) 722. 8. Plattner, H, Wolfram, D, Bachmann, L & Wachter, E. Histochemistry 45 (.1975)1. 9. Sonneborn, T M. J exp zoo1 113 (1950) 87. IO. Plattner, H & Fuchs, S, Histochemisrry 44 (1975) 23.
11. Plattner, H, Wachter, E & Grobner, P. In preparation. 12. Beisson, J, Lefort-Tran. M, Pouphile, M. Rossignal, M & Satir, B, J cell bio169 (1976) 126. 13. Jost, P, Brooks, U J & Griffith. 0 H. 5 mol biol 76 (1973) 313. 14. Wyroba, E & Przelecka, A, Z zellforsch mikroskop anat 143 (1973) 343. 15. Luft, J H, Anat ret 171 (1971) 347. 16. Hausmann, K & Allen, R D, J cell biol 69 (1976) 313.
Received August 6, 1976 Accepted August 24. 1976
The growth of mouse neuroblastoma cells in controlled orientations on thin films of silicon monoxide A. COOPER, H. R. MUNDEN and 6. L. BROWN. Medicul Research Council Cell Biophysics Unit, Biophysics Department, King’s College. University qf London, London, WC2B 5RL, UK Summary. Mouse neuroblastoma cells bave been grown in orientated arrays on fine lines of silicon monoxide evaporated onto plastic Petri dishes. The axon-like orocesses produced by these cells on attachment to this surfaci have also been found to grow along the silicon monoxide lines, giving chains of neuroblastoma cells connected by processes.
Augusti-Tocco & Sato [l] first established clonal cell lines from a transplantable mouse neuroblastoma, C1300. They demonstrated that the cells grew as round cells in suspension in cultures in plastic Petri dishes, with surfaces to which they did not attach, but in plastic tissue culture dishes with treated surfaces the cells attached, flattened and grew neurites, long processes like axons. Many other clones have since been established by them and other workers which
436 Preliminary notes
Fig. 1. Mouse neuroblastoma cells,. clone NB41A. after 3 days’ growth on an array of lines of silicon monoxide, 0.004 mm wide, 50 nm thick, deposited on Falcon plastic Petri dishes. x40. Exp Ceil Res 103 (1976)
Fig. 2. Mouse neuroblastoma cells clone NB4lA growing on a small region of one of the lines of the plate shown in fig. 1, after 3 days’ growth. X 100.
have been shown to have many of the properties of neurons such as chemically and electrically excitable membranes, and the ability to metabolize neurotransmitters [2-51. Recently, the formation of synaptic junctions between neuroblastoma ceils and muscle cell< in culture has been observed
L61. The purpose of the experiments to be described was to obtain ordered arrays of neuroblastoma cells connected by processes growing in a controlled orientation for the study of intercellular interactions, the possible formation of synapses in culture and of microcircuits containing arrays of neurobiastoma cells. Marerials and Methods The mouse neuroblastoma cells used were of clone NB41A, supplied by Dr Gabriella Augusti-Tocco, which were grown in Ham’s FlO medium with 10% fetal calf serum in a 5 % CO,/95 % air mixture at 36°C in Falcon tissue culture dishes. For the oriented growth experiments Falcon polystyrene Petri dishes. diameter 60 MM, with non-adherent surfaces to which the neuroblastoma cells do not attach, were used. An array of fine lines of silicon monoxide, in the form of radial spokes, 0.004 mm, wide and about 50 nm thick, was applied to the internal base surface of a Petri dish by using a masking technique and high vacuum deposition. A Petri dish was mounted on a rotary stage inside the vacuum work chamber and the mask containing a fine line aperture, 0.004 mm, wide by 45 mm long. was precisely located within the dish against the internal surface. The silicon monoxide was evaporated from a tungsten spiral 12 cm from the dish. Following the deposition of the first line, the mask was raised and the dish rotated through 45” and the mask relocated and another line of silicon monoxide deuosited without breaking the vacuum. The sequence -was continued until eight radial lines had been deposited. To observe the cells at high magnification using an oil immersion objective (1.3 N.A.) the base thickness of some of the plastic Petri dishes was reduced from approx. 0.5 mm to about 0.015 mm in the central region by machining with a cutting tool refrigerated by a thermoelectric module. and the surface repolished. The treated Petri dishes were sterilised with 70% alcohol overnight and 1-2~ lo5 mouse neuroblastoma ceils were seeded into 5 ml of Ham’s FIO medium with 10% foetal calf serum in the dish and the cells distributed by swirling. After incubation at 36°C in an atmosphere of 5 % CO,/95 % air overnight, the medium and unattached cells were removed by suction and the medium replaced. The culture was incubated as before for one month, changes of medium being made as required.
Results Fig. 1 shows mouse neuroblastoma ceils growing on a plastic Petri dish with an array of silicon monoxide lines after 3 days incubation. The neuroblastoma cells are clearly adhering to and growing on the sihcon monoxide lines. Eight hours after inoculation, processes were seen to be aligned along the silicon monoxide tracts. After one week some cells began to float off the hnes due apparently to overpopulation of t lines. These cells were removed during medium changes. Removal of these excess cells allowed the development of more processes and the extension of existing ones. The extension of the processes took place along the line of the silicon monoxide with a preference for each edge between the plastic surface and the silicon monoxide as shown in fig. 2. The ceil processes graw in a bipolar fashion from the cell body, which is less flattened than in cultures on Falcon tissue culture dishes as shown in fig, 3. where the complexity of the growth of processes unconstricted by the nature of the surface is evident. After one month in culture, well-defined chains of cells connected together with cesses which were stable with time and connected physically, as tested by pulling with microneedles, were present as shown jn fig. 4.
Several methods have been described for controlling the growth and movement of mammalian cells in tissue culture dishes by modifying the surface, including metal deposition [7, 81, coating the surface with proteins [9, lo] and phospholipids [I 11.The work of Weiss [12], Letourneau [13], Bunge [14] and Bray [15] shows that interaction between the plasma membrane at the point
Fig. 3. Mouse neuroblastoma cells, clone NB41A, after 3 days’ growth on Falcon tissue culture plates under similar culture conditions as for the cells shown in fig. 1. x 100.
Fig.
of cellular contact or at the axon or neurite tip, with the neighbouring surface is an important determinant of the direction of cell locomotion, aggregation or direction of growth of the process or tip. Thin films of silicon monoxide appear to provide a suitable surface with which the plasma membranes of neuroblastoma cells interact so that the cells attach and grow processes
which grow in patterns controlled by the boundaries of the films. The discovery that silicon monoxide films provide such a surface is a very useful one as an extensive technology using films of this material has been developed for manufacturing microelectronic circuits which can be used to prepare patterns for growing neurobiastoma cells and possibly
Exp CellRes 103(1976)
4. Mouse neuroblastoma cells clone NB41A growing on a small region of a plate similar to that described in fig. 1 but with thin central observing area, after one month in culture. x800.
Preliminary ?;otes 439 other cells in given arrays. These arrays could. be used for the study of the formation of synapses between clones of neuroblastoma cells and other cells originating from nervous tissue and for the study of the electrical properties of defined microcircuits containing such cells.
The folded-fiber structure of human ciaromosomes and interchromosomal fibers has been demonstrated by whole-mount transmission [3, 7] and scanning [5] electron microscopy. Although the fibrous structure [ 1, 41 and interchromosomal connections [2] have also been shown in conventional acid-alcohol fixed and air-dried preparaWe wish to thank Mr Z. Gabot for the photographs. tions of human chromosomes by electron microscopy, they have not yet been shown References distinctly by light microscopy. I. August-Tocco, G & Sato. G, Proc natl acad sci Immunoperoxidase technique is useful US 64 (1969) 311. 2. Nelson, P, Ruffner, W & Nirenberg, M, Proc natl for the study of chromosomal structure acad sci US 64 (1969) 1004. 3. Harris, A J & Dennis, M J, Science 167 (1970) when certain specific antibodies are use 1253. [6, 81. By utilizing this technique, we 4. Peacock, J, Minna. J, Nelson. P & Nirenberg, M, stained human chromosomes using antiExp cell res 73 (1972) 367. 5. Schubert, D, Humphreys, S, Baroni, C & Cohn, native DNA antibody obtained from a paM, Proc natl acad sci US 64 (1969) 316. 6. Amano, T, Richelson, E & Nirenberg. M, Proc tient with systemic lupus erythematosuc natl acad sci US 69 (1972) 258. (SLE), and observed the fibrous structure 7. Carter. S El, Nature 208 (1965) 1183. and interchromosomal connections in con8. Letoumeau, P C, Dev bio144 (1975) 77. 9. Witkowski, J A & Brighton. W D. Exp cell res ventional air-dried preparations with a light 70 (1972) 41. 10. Miller, C A & Levine, E M,‘Science 177 (1972) microscope. 799. Il. Ivanova. 0 Y & Margolis, L B, Nature 242 (1973) 200. 12. Weiss. P, Exp cell res, suppl. 8 (1961) 260. 13. Letourneau, P C, Dev biol44 (1975) 92. 14. Bunge, MB, J cell biol 56 (1973) 713. 15. Bray, D, Proc natl acad sci US 65 (1970) 905. Received August 3, 1976 .4ccepted September 17. 1976
Interchromosomal connections as revealed by an immunoperoxidase technique applying anti-DNA antibody Y. KANAYAMA, K. YAMAKAMI, T. INOUE, Y. MAEDA and K. SIOTA, First Department of Internal Medicine, Osaka School, Osaka, 545, Japan
City
University
Medical
Summary. An immunoperoxidase technique was applied to the study of chromosomal structure. Using the immunoperoxidase technique with the anti-native DNA antibody, fibrous structure of human chromosomes and interchromosomal connections could be revealed by light microscopy in acid-alcohol fixed and air-dried chromosomal preparations. 29-761811
Materials atzd Methods As a source of anti-DNA antibody. we used serum from a patient (H. K.) with SLE. which cvas tested bv the double diffusion method in agarose [ 121,producing a precipitin line with native DNA from calf thymus (Sigma), but not with heat-denatured DNA or calf thymus histone (Sigma). Chromosomal preparations were made from phytohemagglutinin (PHA)-stimulated human peripheral lymphocytes, after hypotonic treatment with 0.075 M KC1 for 10 min and fixation with 3 : 1 methanol/acetic acid. The chromosomes were then spread on slides. air-dried, stored at 4°C and used within a week. The indirect immunoperoxidase technique was anplied with slight modifications as described by Nakane & Pierce r91. The slide preuarations were allowed to react withthe anti-DNAantibody (the serum of H. K.) for 15 min in a moist chamber at room temperature. After being rinsed briefly with phosphate-buffered saline solution (PBS) of PH 7.2. the prenared slides were allowed to react for 15 min with* pgroxidase-labelled anti-human IgG (Dakkopatts), which was diluted i : 2o with PBS. They were again rinsed with PBS, then incubated for 8 min at room temoerature in a solution of 20 mg of diaminobenzidine (4.HCl) and 0.005% peroxide in 100 ml of 0.05 M Tris buffer solution (PI-I 4.6). The dides were then washed with distilled water, dehydrated in graded alcohol. cleared in xylol and mounted. Sera from healthy persons and the serum (H. K.) absorbed with native DNA were used for control staining and some preparations were stained with
rived from these labelling experiments is that, at the exocytosis sites, cell membrane materials flow in a centropetal direction; this mechanism allows for subsequent membrane resealing. Results are summarized in fig. 4 (A-G): Upon Ca*+-influx (B, [lo]) the apical trichocyst membrane and the cell membrane portion encircled by a “ring” undergo fragmentation (C), allowing the trichocyst contents to be discharged (0). At this stage the cell membrane and the trichocyst membrane have fused with each other within “rings” (II). Subsequently the exocytosis channels become narrower as some cell membrane and underlying cytoplasmic materials spread to within the “rings” (E). Some surface label spreads from the cell membrane onto the uppermost trichocyst membrane region (F). As the labelling density of the cell membrane around exocytosis sites appears “undiluted” at all stages, this indicates a centropetal diffusion of cell membrane materials, which then allows the cell membrane and the trichocyst membrane to become completely resealed and to become finally detached from each other (G). All this evidence leads to the conclusion that with Paramecium cells exocytosis involves retrieval of the secretory vesicle membranes rather than their integration into the cell membrane. This conclusion is in agreement with a recent morphological analysis by Hausmann & Allen [ 161. This study was supported by grant no. 2923 from the “Osterreichische Fonds zur Forderung der wissenschaftlichen Forschung”.
Rqferences 1. Winkler, H, Phil tram roy sot London B 261(1971) 293.
2. Satir, B, Schooley, C & Satir, P. J cell biol 56 (1973) 153. 3. Satir, B, Sci Am 233 (1975) 28.
4. Van Wagtendonk, W J, Paramecium. A current survey. Elsevier, Amsterdam, London, New York (19743.
mxes
435
5. Bachmann, L. Schmitt, W W Bi Plattner, H, Proc 5th Em congr electron microscopy, Manchester (ed V E Cosslett) D. 244. The Institute of Phvsics, . London, Bristol (f972). 6. Plattner. H, Miller, F & Bachmann, L. J cell sci !3 (1973) 687. 7. Plattner, H, Nature 252 (1974) 722. 8. Plattner, H, Wolfram, D, Bachmann, L & Wachter, E. Histochemistry 45 (.1975)1. 9. Sonneborn, T M. J exp zoo1 113 (1950) 87. IO. Plattner, H & Fuchs, S, Histochemisrry 44 (1975) 23.
11. Plattner, H, Wachter, E & Grobner, P. In preparation. 12. Beisson, J, Lefort-Tran. M, Pouphile, M. Rossignal, M & Satir, B, J cell bio169 (1976) 126. 13. Jost, P, Brooks, U J & Griffith. 0 H. 5 mol biol 76 (1973) 313. 14. Wyroba, E & Przelecka, A, Z zellforsch mikroskop anat 143 (1973) 343. 15. Luft, J H, Anat ret 171 (1971) 347. 16. Hausmann, K & Allen, R D, J cell biol 69 (1976) 313.
Received August 6, 1976 Accepted August 24. 1976
The growth of mouse neuroblastoma cells in controlled orientations on thin films of silicon monoxide A. COOPER, H. R. MUNDEN and 6. L. BROWN. Medicul Research Council Cell Biophysics Unit, Biophysics Department, King’s College. University qf London, London, WC2B 5RL, UK Summary. Mouse neuroblastoma cells bave been grown in orientated arrays on fine lines of silicon monoxide evaporated onto plastic Petri dishes. The axon-like orocesses produced by these cells on attachment to this surfaci have also been found to grow along the silicon monoxide lines, giving chains of neuroblastoma cells connected by processes.
Augusti-Tocco & Sato [l] first established clonal cell lines from a transplantable mouse neuroblastoma, C1300. They demonstrated that the cells grew as round cells in suspension in cultures in plastic Petri dishes, with surfaces to which they did not attach, but in plastic tissue culture dishes with treated surfaces the cells attached, flattened and grew neurites, long processes like axons. Many other clones have since been established by them and other workers which
436 Preliminary notes
Fig. 1. Mouse neuroblastoma cells,. clone NB41A. after 3 days’ growth on an array of lines of silicon monoxide, 0.004 mm wide, 50 nm thick, deposited on Falcon plastic Petri dishes. x40. Exp Ceil Res 103 (1976)
Fig. 2. Mouse neuroblastoma cells clone NB4lA growing on a small region of one of the lines of the plate shown in fig. 1, after 3 days’ growth. X 100.
have been shown to have many of the properties of neurons such as chemically and electrically excitable membranes, and the ability to metabolize neurotransmitters [2-51. Recently, the formation of synaptic junctions between neuroblastoma ceils and muscle cell< in culture has been observed
L61. The purpose of the experiments to be described was to obtain ordered arrays of neuroblastoma cells connected by processes growing in a controlled orientation for the study of intercellular interactions, the possible formation of synapses in culture and of microcircuits containing arrays of neurobiastoma cells. Marerials and Methods The mouse neuroblastoma cells used were of clone NB41A, supplied by Dr Gabriella Augusti-Tocco, which were grown in Ham’s FlO medium with 10% fetal calf serum in a 5 % CO,/95 % air mixture at 36°C in Falcon tissue culture dishes. For the oriented growth experiments Falcon polystyrene Petri dishes. diameter 60 MM, with non-adherent surfaces to which the neuroblastoma cells do not attach, were used. An array of fine lines of silicon monoxide, in the form of radial spokes, 0.004 mm, wide and about 50 nm thick, was applied to the internal base surface of a Petri dish by using a masking technique and high vacuum deposition. A Petri dish was mounted on a rotary stage inside the vacuum work chamber and the mask containing a fine line aperture, 0.004 mm, wide by 45 mm long. was precisely located within the dish against the internal surface. The silicon monoxide was evaporated from a tungsten spiral 12 cm from the dish. Following the deposition of the first line, the mask was raised and the dish rotated through 45” and the mask relocated and another line of silicon monoxide deuosited without breaking the vacuum. The sequence -was continued until eight radial lines had been deposited. To observe the cells at high magnification using an oil immersion objective (1.3 N.A.) the base thickness of some of the plastic Petri dishes was reduced from approx. 0.5 mm to about 0.015 mm in the central region by machining with a cutting tool refrigerated by a thermoelectric module. and the surface repolished. The treated Petri dishes were sterilised with 70% alcohol overnight and 1-2~ lo5 mouse neuroblastoma ceils were seeded into 5 ml of Ham’s FIO medium with 10% foetal calf serum in the dish and the cells distributed by swirling. After incubation at 36°C in an atmosphere of 5 % CO,/95 % air overnight, the medium and unattached cells were removed by suction and the medium replaced. The culture was incubated as before for one month, changes of medium being made as required.
Results Fig. 1 shows mouse neuroblastoma ceils growing on a plastic Petri dish with an array of silicon monoxide lines after 3 days incubation. The neuroblastoma cells are clearly adhering to and growing on the sihcon monoxide lines. Eight hours after inoculation, processes were seen to be aligned along the silicon monoxide tracts. After one week some cells began to float off the hnes due apparently to overpopulation of t lines. These cells were removed during medium changes. Removal of these excess cells allowed the development of more processes and the extension of existing ones. The extension of the processes took place along the line of the silicon monoxide with a preference for each edge between the plastic surface and the silicon monoxide as shown in fig. 2. The ceil processes graw in a bipolar fashion from the cell body, which is less flattened than in cultures on Falcon tissue culture dishes as shown in fig, 3. where the complexity of the growth of processes unconstricted by the nature of the surface is evident. After one month in culture, well-defined chains of cells connected together with cesses which were stable with time and connected physically, as tested by pulling with microneedles, were present as shown jn fig. 4.
Several methods have been described for controlling the growth and movement of mammalian cells in tissue culture dishes by modifying the surface, including metal deposition [7, 81, coating the surface with proteins [9, lo] and phospholipids [I 11.The work of Weiss [12], Letourneau [13], Bunge [14] and Bray [15] shows that interaction between the plasma membrane at the point
Fig. 3. Mouse neuroblastoma cells, clone NB41A, after 3 days’ growth on Falcon tissue culture plates under similar culture conditions as for the cells shown in fig. 1. x 100.
Fig.
of cellular contact or at the axon or neurite tip, with the neighbouring surface is an important determinant of the direction of cell locomotion, aggregation or direction of growth of the process or tip. Thin films of silicon monoxide appear to provide a suitable surface with which the plasma membranes of neuroblastoma cells interact so that the cells attach and grow processes
which grow in patterns controlled by the boundaries of the films. The discovery that silicon monoxide films provide such a surface is a very useful one as an extensive technology using films of this material has been developed for manufacturing microelectronic circuits which can be used to prepare patterns for growing neurobiastoma cells and possibly
Exp CellRes 103(1976)
4. Mouse neuroblastoma cells clone NB41A growing on a small region of a plate similar to that described in fig. 1 but with thin central observing area, after one month in culture. x800.
Preliminary ?;otes 439 other cells in given arrays. These arrays could. be used for the study of the formation of synapses between clones of neuroblastoma cells and other cells originating from nervous tissue and for the study of the electrical properties of defined microcircuits containing such cells.
The folded-fiber structure of human ciaromosomes and interchromosomal fibers has been demonstrated by whole-mount transmission [3, 7] and scanning [5] electron microscopy. Although the fibrous structure [ 1, 41 and interchromosomal connections [2] have also been shown in conventional acid-alcohol fixed and air-dried preparaWe wish to thank Mr Z. Gabot for the photographs. tions of human chromosomes by electron microscopy, they have not yet been shown References distinctly by light microscopy. I. August-Tocco, G & Sato. G, Proc natl acad sci Immunoperoxidase technique is useful US 64 (1969) 311. 2. Nelson, P, Ruffner, W & Nirenberg, M, Proc natl for the study of chromosomal structure acad sci US 64 (1969) 1004. 3. Harris, A J & Dennis, M J, Science 167 (1970) when certain specific antibodies are use 1253. [6, 81. By utilizing this technique, we 4. Peacock, J, Minna. J, Nelson. P & Nirenberg, M, stained human chromosomes using antiExp cell res 73 (1972) 367. 5. Schubert, D, Humphreys, S, Baroni, C & Cohn, native DNA antibody obtained from a paM, Proc natl acad sci US 64 (1969) 316. 6. Amano, T, Richelson, E & Nirenberg. M, Proc tient with systemic lupus erythematosuc natl acad sci US 69 (1972) 258. (SLE), and observed the fibrous structure 7. Carter. S El, Nature 208 (1965) 1183. and interchromosomal connections in con8. Letoumeau, P C, Dev bio144 (1975) 77. 9. Witkowski, J A & Brighton. W D. Exp cell res ventional air-dried preparations with a light 70 (1972) 41. 10. Miller, C A & Levine, E M,‘Science 177 (1972) microscope. 799. Il. Ivanova. 0 Y & Margolis, L B, Nature 242 (1973) 200. 12. Weiss. P, Exp cell res, suppl. 8 (1961) 260. 13. Letourneau, P C, Dev biol44 (1975) 92. 14. Bunge, MB, J cell biol 56 (1973) 713. 15. Bray, D, Proc natl acad sci US 65 (1970) 905. Received August 3, 1976 .4ccepted September 17. 1976
Interchromosomal connections as revealed by an immunoperoxidase technique applying anti-DNA antibody Y. KANAYAMA, K. YAMAKAMI, T. INOUE, Y. MAEDA and K. SIOTA, First Department of Internal Medicine, Osaka School, Osaka, 545, Japan
City
University
Medical
Summary. An immunoperoxidase technique was applied to the study of chromosomal structure. Using the immunoperoxidase technique with the anti-native DNA antibody, fibrous structure of human chromosomes and interchromosomal connections could be revealed by light microscopy in acid-alcohol fixed and air-dried chromosomal preparations. 29-761811
Materials atzd Methods As a source of anti-DNA antibody. we used serum from a patient (H. K.) with SLE. which cvas tested bv the double diffusion method in agarose [ 121,producing a precipitin line with native DNA from calf thymus (Sigma), but not with heat-denatured DNA or calf thymus histone (Sigma). Chromosomal preparations were made from phytohemagglutinin (PHA)-stimulated human peripheral lymphocytes, after hypotonic treatment with 0.075 M KC1 for 10 min and fixation with 3 : 1 methanol/acetic acid. The chromosomes were then spread on slides. air-dried, stored at 4°C and used within a week. The indirect immunoperoxidase technique was anplied with slight modifications as described by Nakane & Pierce r91. The slide preuarations were allowed to react withthe anti-DNAantibody (the serum of H. K.) for 15 min in a moist chamber at room temperature. After being rinsed briefly with phosphate-buffered saline solution (PBS) of PH 7.2. the prenared slides were allowed to react for 15 min with* pgroxidase-labelled anti-human IgG (Dakkopatts), which was diluted i : 2o with PBS. They were again rinsed with PBS, then incubated for 8 min at room temoerature in a solution of 20 mg of diaminobenzidine (4.HCl) and 0.005% peroxide in 100 ml of 0.05 M Tris buffer solution (PI-I 4.6). The dides were then washed with distilled water, dehydrated in graded alcohol. cleared in xylol and mounted. Sera from healthy persons and the serum (H. K.) absorbed with native DNA were used for control staining and some preparations were stained with
