- Email: [email protected]
Fixation and embedding of mammalian brain and spinal cord for electron microscopy
108
J. ULTRASTRUCTURE RESEARCH
4,
108-112
(1960)
Fixation and Embedding of Mammalian Brain and Spinal Cord for Electron Microscopy SARAHA. LUSL, M.D. Department of Pathology and Anatomy, and the Beaumont May Institute of Neurology, Washington University School of Medicine, St. Louis, Missouri Received March 23, 1960
White matter of the brain was fixed in 1% osmium tetroxide (Dalton's fixative) to which was added saponin. It was found that adding saponin to the fixative, and following the gradual dehydration by a toluene step greatly improved preservation of myelin of the central nervous system. The technic of tissue preparation for electron microscopy has been in an almost constant state of flux since fixation in buffered osmium tetroxide was reported by Palade (8) and plastic embedding by Newman, Boryski, and Swerdlow (7). The nervous system in particular has presented unique difficulties in both fixation and embedding that have hinged on the presence of myelin and the maintenance of its integrity. Difficult as it has been to study the fine structure of normal brain or cord, because of the artifactitious separation of myelin lamellae, at least it has been possible to distinguish that which was normal from artifact. However, observations on the abnormal myelin sheaths in demyelinating diseases will be virtually meaningless if the integrity of uninjured myelin is open to question. In abnormalities of myelin, defects in the forces holding the lamellae together may be an early manifestation of disease, which can be evaluated only if the normal myelin sheath is uniformly and adequately preserved. Important fundamental contributions concerning the structural organization of myelin and its reactions under varying physical conditions have been made in recent years. Finean and Millington (5) examined peripheral nerve myelin as to its alterations in hypotonic and hypertonic immersion media. They observed no evidence of free water between the myelin layers since no contraction occurred in hypertonic solutions. In hypotonic solutions, however, there was a rapid expansion of the layers. More recently, Fern~mdez-Mor(tn and Finean (3) and Finean (4) have examined the low angle X-ray diffraction patterns of both peripheral and central myelin in relation to various fixatives and embedding media. Their findings are of particular interest because they have shown that there is a shrinking of the layer spacing during dehydration in graded alcohols and that, with immersion in methacrylate, there is a rapid expansion again to a pattern closely resembling that of moist osmium tetroxide fixed
FIa. 1. Low-power electron micrograph of optic nerve of adult mouse. Myelin sheaths of both large and small axons are intact throughout the relatively large field. Only scant glial processes separate the myelinated axons in this portion of the optic nerve, x 3750. FIG. 2. Slightly higher magnification of another area from an optic nerve of an adult mouse, x 7500.
110
SARAH A. LUSE
myelin. Furthermore, they have observed additional expansion of myelin structure during polymerization, as well as a deterioration in definition of the diffraction pattern. A variety of fixatives have been utilized in the preparation of brain and spinal cord for electron microscopy. Veronal acetate buffered osmium tetroxide has certain advantages in clarity of detail of membranes, but does not lead to satisfactory preservation of the myelin sheaths of the white matter of either the brain or spinal cord. Potassium permanganate (6, 10), which has been used effectively on peripheral myelinated axons, so alters the appearance of the various complexly intertwined components of the central nervous system that interpretation is made difficult if not impossible. Dalton's chrome-osmium tetroxide fixative (2) has so far proved the most satisfactory for preserving all structures in brain. Ward (11) has suggested addition of uranyl acetate to the methacrylate embedding material as a means of reducing splitting of myelin. Newer embedding media, the epozy resins (1), also reduce the distortion of layered membrane systems. They do not, however, lend themselves to the preparation of the large number of blocks necessary for the study of vaguely localized diseases of the nervous system, nor are we yet certain that epoxy resins will maintain the same consistency for cutting over a long period of storage. We have fixed white matter in Dalton's chrome-osmium tetroxide solution followed by dehydration in small (10%) increments of ethanol, partial prepolymerization of the methacrylate and subsequent polymerization at 60°C. Although this technic reduces disruption of myelin, it has been impossible to obtain uniformly intact myelin sheaths around all axons. For the most part isolated myelinated fibers in the cortex and small myelinated fibers in the white matter have had intact sheaths, whereas many of the larger axons of the white matter have been surrounded by distorted myelin. Oddly enough, it is far easier to obtain intact myelin about axons of a peripheral nerve than about central axons. Integrity of the myelin sheaths in white matter can be obtained with a high degree of uniformity in methacrylate-embedded tissue by the addition of saponin to the chrome-osmium fixative, and by clearing in toluene (I, 9) prior to infiltration in methacrylate. The mechanisms responsible for the integrity of myelin following these procedures are unknown. It has been noted that use of the toluene step and omission of saponin in the fixative leads to relatively adequate maintenance of the myelin, but in our hands use of both has been more satisfactory. FIo. 3. Electron micrograph of cerebrum of adult rabbit to demonstrate at low magnification the myelin sheaths which are enlarged in Figs. 4 and 5. x 10,000. FIo. 4. Higher magnification of a portion of Fig. 3 to show the maintenance of the lamellar structure of the myelin, x 135,000 approximately. FIG. 5. Another field to demonstrate maintenance of myelin lamellae, x 105,000 approximately.
liiii~ii!ii
ii~iii!
i~~¸ i~
112
SARAH A. LUSE
Technic: (1) Fix for 1 hr in a 1% solution of osmium tetroxide (Dalton's) at pH 7.4 to which has been added two drops of a 1% aqueous solution of saponin per 3 ml of fixative. (Purified Saponin, J. T. Baker Chemical Co., Phillipsbury, N.Y.) (2) Dehydrate with ten-minute changes in graded concentrations of ethanol (10%, 20%, 30%, etc. to 95%). (3) Two changes of absolute ethanol, 15 rain each. (4) Ten minutes in one part toluene: one part absolute ethanol. (5) Thirty minutes in toluene. (6) Ten minutes in one part toluene; one part methacrylate. (7) Two changes of 7 : 1 butyl-methyl methacrylate, 30 rain each. (8) Thirty minutes in 7 : 1 methacrylate plus 0.2% benzoyl peroxide. (9) Embed in gelatine capsules filled with a partially polymerized mixture of methacrylate and benzoyl peroxide and place in oven overnight at 60°C. The extent to which this procedure of fixation, dehydration and embedding preserves myelin sheaths in the white matter of the brain is demonstrated in Figs. 1, 2, and 3, where at low magnification it is evident that the sheaths of all axons within the field are essentially intact. Figs. 4 and 5 illustrate the appearance of these myelin sheaths at somewhat higher magnifications in order to demonstrate their lamellar character. The mechanisms involved in the addition of saponin, a surface-wetting agent and a glycoside, and of toluene, a clearing and hardening agent, are unknown; but their empirical use is justified by the improvement in the preservation of white matter. This is important in studying the normal prior to the extension of electron microscopic studies to diseases of the central nervous system. This work was supported in part by United States Public Health Service Grants B1539 and B425. I am indebted to Keith C. Richardson for his suggestions and help in this work.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
BrRBECK, M. S. and MERCER,E. H., J. Roy. Microscop. Soc. 76, 168 (1957). DALTON,A. J., Anat. Rec. 121, 281 (1955). FERN,g,NDEZ-MOR~N, H. and FINEAN, J. B., J. Biophys. Biochem. Cytol. 3, 725 (1957). FINlSAN,J. B., Exp. Cell Res., Suppl. 5, 18 (1957). FINEAN, J. B. and MILLINGTON,P. F., dr. Biophys. Biochem. Cytol. 3, 89 (1957). LtrET, J. H., J. Biophys. Biochem. Cytol. 2, 799 (1956). NEWMAN, S. V., BORYSKO,E. and SWERDLOW, M., Science 110, 66 (1949). PALADE, G. E., or. exp. Med. 9, 285 (1952). RICHARDSON,K. C., JARRET, L. and FINKE, E. H. In press. ROBERTSON,J. D., J. Biophys. Biochem. Cytol. 4, 349 (1958). WARD, R. T., J. Histochem. Cytochem. 6, 398 (1958).
J. ULTRASTRUCTURE RESEARCH
4,
108-112
(1960)
Fixation and Embedding of Mammalian Brain and Spinal Cord for Electron Microscopy SARAHA. LUSL, M.D. Department of Pathology and Anatomy, and the Beaumont May Institute of Neurology, Washington University School of Medicine, St. Louis, Missouri Received March 23, 1960
White matter of the brain was fixed in 1% osmium tetroxide (Dalton's fixative) to which was added saponin. It was found that adding saponin to the fixative, and following the gradual dehydration by a toluene step greatly improved preservation of myelin of the central nervous system. The technic of tissue preparation for electron microscopy has been in an almost constant state of flux since fixation in buffered osmium tetroxide was reported by Palade (8) and plastic embedding by Newman, Boryski, and Swerdlow (7). The nervous system in particular has presented unique difficulties in both fixation and embedding that have hinged on the presence of myelin and the maintenance of its integrity. Difficult as it has been to study the fine structure of normal brain or cord, because of the artifactitious separation of myelin lamellae, at least it has been possible to distinguish that which was normal from artifact. However, observations on the abnormal myelin sheaths in demyelinating diseases will be virtually meaningless if the integrity of uninjured myelin is open to question. In abnormalities of myelin, defects in the forces holding the lamellae together may be an early manifestation of disease, which can be evaluated only if the normal myelin sheath is uniformly and adequately preserved. Important fundamental contributions concerning the structural organization of myelin and its reactions under varying physical conditions have been made in recent years. Finean and Millington (5) examined peripheral nerve myelin as to its alterations in hypotonic and hypertonic immersion media. They observed no evidence of free water between the myelin layers since no contraction occurred in hypertonic solutions. In hypotonic solutions, however, there was a rapid expansion of the layers. More recently, Fern~mdez-Mor(tn and Finean (3) and Finean (4) have examined the low angle X-ray diffraction patterns of both peripheral and central myelin in relation to various fixatives and embedding media. Their findings are of particular interest because they have shown that there is a shrinking of the layer spacing during dehydration in graded alcohols and that, with immersion in methacrylate, there is a rapid expansion again to a pattern closely resembling that of moist osmium tetroxide fixed
FIa. 1. Low-power electron micrograph of optic nerve of adult mouse. Myelin sheaths of both large and small axons are intact throughout the relatively large field. Only scant glial processes separate the myelinated axons in this portion of the optic nerve, x 3750. FIG. 2. Slightly higher magnification of another area from an optic nerve of an adult mouse, x 7500.
110
SARAH A. LUSE
myelin. Furthermore, they have observed additional expansion of myelin structure during polymerization, as well as a deterioration in definition of the diffraction pattern. A variety of fixatives have been utilized in the preparation of brain and spinal cord for electron microscopy. Veronal acetate buffered osmium tetroxide has certain advantages in clarity of detail of membranes, but does not lead to satisfactory preservation of the myelin sheaths of the white matter of either the brain or spinal cord. Potassium permanganate (6, 10), which has been used effectively on peripheral myelinated axons, so alters the appearance of the various complexly intertwined components of the central nervous system that interpretation is made difficult if not impossible. Dalton's chrome-osmium tetroxide fixative (2) has so far proved the most satisfactory for preserving all structures in brain. Ward (11) has suggested addition of uranyl acetate to the methacrylate embedding material as a means of reducing splitting of myelin. Newer embedding media, the epozy resins (1), also reduce the distortion of layered membrane systems. They do not, however, lend themselves to the preparation of the large number of blocks necessary for the study of vaguely localized diseases of the nervous system, nor are we yet certain that epoxy resins will maintain the same consistency for cutting over a long period of storage. We have fixed white matter in Dalton's chrome-osmium tetroxide solution followed by dehydration in small (10%) increments of ethanol, partial prepolymerization of the methacrylate and subsequent polymerization at 60°C. Although this technic reduces disruption of myelin, it has been impossible to obtain uniformly intact myelin sheaths around all axons. For the most part isolated myelinated fibers in the cortex and small myelinated fibers in the white matter have had intact sheaths, whereas many of the larger axons of the white matter have been surrounded by distorted myelin. Oddly enough, it is far easier to obtain intact myelin about axons of a peripheral nerve than about central axons. Integrity of the myelin sheaths in white matter can be obtained with a high degree of uniformity in methacrylate-embedded tissue by the addition of saponin to the chrome-osmium fixative, and by clearing in toluene (I, 9) prior to infiltration in methacrylate. The mechanisms responsible for the integrity of myelin following these procedures are unknown. It has been noted that use of the toluene step and omission of saponin in the fixative leads to relatively adequate maintenance of the myelin, but in our hands use of both has been more satisfactory. FIo. 3. Electron micrograph of cerebrum of adult rabbit to demonstrate at low magnification the myelin sheaths which are enlarged in Figs. 4 and 5. x 10,000. FIo. 4. Higher magnification of a portion of Fig. 3 to show the maintenance of the lamellar structure of the myelin, x 135,000 approximately. FIG. 5. Another field to demonstrate maintenance of myelin lamellae, x 105,000 approximately.
liiii~ii!ii
ii~iii!
i~~¸ i~
112
SARAH A. LUSE
Technic: (1) Fix for 1 hr in a 1% solution of osmium tetroxide (Dalton's) at pH 7.4 to which has been added two drops of a 1% aqueous solution of saponin per 3 ml of fixative. (Purified Saponin, J. T. Baker Chemical Co., Phillipsbury, N.Y.) (2) Dehydrate with ten-minute changes in graded concentrations of ethanol (10%, 20%, 30%, etc. to 95%). (3) Two changes of absolute ethanol, 15 rain each. (4) Ten minutes in one part toluene: one part absolute ethanol. (5) Thirty minutes in toluene. (6) Ten minutes in one part toluene; one part methacrylate. (7) Two changes of 7 : 1 butyl-methyl methacrylate, 30 rain each. (8) Thirty minutes in 7 : 1 methacrylate plus 0.2% benzoyl peroxide. (9) Embed in gelatine capsules filled with a partially polymerized mixture of methacrylate and benzoyl peroxide and place in oven overnight at 60°C. The extent to which this procedure of fixation, dehydration and embedding preserves myelin sheaths in the white matter of the brain is demonstrated in Figs. 1, 2, and 3, where at low magnification it is evident that the sheaths of all axons within the field are essentially intact. Figs. 4 and 5 illustrate the appearance of these myelin sheaths at somewhat higher magnifications in order to demonstrate their lamellar character. The mechanisms involved in the addition of saponin, a surface-wetting agent and a glycoside, and of toluene, a clearing and hardening agent, are unknown; but their empirical use is justified by the improvement in the preservation of white matter. This is important in studying the normal prior to the extension of electron microscopic studies to diseases of the central nervous system. This work was supported in part by United States Public Health Service Grants B1539 and B425. I am indebted to Keith C. Richardson for his suggestions and help in this work.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11.
BrRBECK, M. S. and MERCER,E. H., J. Roy. Microscop. Soc. 76, 168 (1957). DALTON,A. J., Anat. Rec. 121, 281 (1955). FERN,g,NDEZ-MOR~N, H. and FINEAN, J. B., J. Biophys. Biochem. Cytol. 3, 725 (1957). FINlSAN,J. B., Exp. Cell Res., Suppl. 5, 18 (1957). FINEAN, J. B. and MILLINGTON,P. F., dr. Biophys. Biochem. Cytol. 3, 89 (1957). LtrET, J. H., J. Biophys. Biochem. Cytol. 2, 799 (1956). NEWMAN, S. V., BORYSKO,E. and SWERDLOW, M., Science 110, 66 (1949). PALADE, G. E., or. exp. Med. 9, 285 (1952). RICHARDSON,K. C., JARRET, L. and FINKE, E. H. In press. ROBERTSON,J. D., J. Biophys. Biochem. Cytol. 4, 349 (1958). WARD, R. T., J. Histochem. Cytochem. 6, 398 (1958).