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Electron microscopy of golgi-stained material following lead chromate substitution
Brain Research, 103 (1976) 339 344 (i~ Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
339
Short Communications
Electron microscopy of golgi-stained material following substitution
lead chromate
E. RAMON-MOLINER AND J. FERRARI D~partement d'Anatomie, Facultd de Mddecine, Universitd de Sherbrooke, Sherbrooke, Que. (Canada)
(Accepted October 23rd, 1975)
The advantages of combining electron microscopy with the study of Golgistained material are now well documented. A significant advance was made by StellS,% Blackstad and Kjaerheim a, and Blackstad 1,2 who demonstrated that direct electron microscopy of previously Golgi-stained material was possible and that a surprising degree of preservation of ultrastructural detail could be achieved despite the chemical insults inflicted upon the tissue. However, direct electron microscopy of non-modified Golgi material has a number of disadvantages and difficulties1,7. For this reason, attempts were made to modify the silver chromate impregnation in order to render it less opaque to the electron beam and facilitate ultramicrotomy. Blackstad 1,~ proved that it was possible to obtain a partial 'de-impregnation' and demonstrated that the Golgi impregnation does not necessarily destroy ultrastructural details. Chan-Palay and Palay 4,a brought the innovation of studying Golgi-stained structures in sections several microns thick by means of high voltage electron microscopy. The present authors 7 have partially transformed the silver chromate precipitate present in Golgistained structures into lead chromate. When this partial replacement was followed by a treatment in a solution containing thiosulphate ions, only lead chromate was left in the tissue. This resulted in a depleted impregnation of the originally Golgi-stained structures without fading of their outlines. By using incident light to study the Araldite-embedded material prior to ultramicrotomy, stained structures appear as bright yellow against the dark background (Figs. I and 2). However, the degree of preservation of the fine ultrastructure was poor and the technique was, in general, difficult to control. The present report presents a method for controlling the degree of lead chromate substitution and improving ultrastructural preservation. One can distinguish the following stages in the performance of the technique: graded substitution, elimination of unwanted lead ions in the background, osmication, removal of unsubstituted silver chromate, dehydration, and embedding in an epoxy resin. In the substitution stage, the sections containing Golgi-stained structures are
340
Fig. I. Fragment of olfactory bulb of rat embedded in Araldite and showing under incident light mitral cells and periglomerular neurons impregnated with lead chromate, x 30. Fig. 2. Trimmed Araldite block showing under incident light the peripheral dendrite of a mitral celt (on the left). The secondary mitral dendrites of the external plexiform layer can be seen on the right. :-~ 120.
341 immersed in a solution of lead lactate buffered with boric acid. Of all the salts tried (nitrate, acetate, chlorate, chloride) lead lactate (K and K Laboratories, Plainview, N.Y. 11803) was the only one that permitted a graded reaction. Lead lactate does not give a massive and nearly instantaneous substitution. At room temperature, no replacement by lead chromate takes place before 10 rain, and at temperatures near 4 °C the reaction is considerably slowed. The presence of small amounts of silver nitrate acts as an inhibitor and for this reason it becomes necessary to change the solution of lead lactate to remove any traces of silver. The concentration of lead lactate does not seem to influence the rate of substitution. When buffered with boric acid, at pH 5, the reaction begins after 15 min. F r o m this moment on, it seems to take place at an accelerated rate so that after half an hour the amount of substitution is excessive. The addition of small amounts of silver nitrate can prolong the substitution time, but the reaction is irregular and difficult to control. With the p H higher than 5, the degree of substitution is lower but this is associated with background turbidity, due to lead precipitation. Once the desired degree of substitution is obtained, it is necessary to stop it without trapping lead in the background. In the absence of lactate ions, which appear to act as a chelating agent, lead is bound to the tissue in such a way that it will react at later stages with the chromate ions liberated by thiocyanate, giving a turbid background and a massive unwanted secondary substitution. Immersing the tissue in a solution containing silver, barium, and lactate ions, at pH 5, arrests the substitution process and washes away all the lead present in the background, without removing the lead chromate already formed within the stained structures. Osmication is carried out in the presence of dichromate ions to prevent excessive darkening of the tissue. Dichromate ions also appear to promote the formation of lead chromate in the stained structures in such a way that it will not diffuse and fade under the action of thiocyanate. The selective removal of non-replaced silver chromate occurs in an acetone solution of a m m o n i u m fluoride and a m m o n i u m thiocyanate. A m m o n i u m fluoride prevents the diffusion of lead chromate, since lead fluoride is highly insoluble in acetone. A m m o n i u m fluoride also can redissolve any metallic silver which may have formed in the background. A m m o n i u m thiocyanate dissolved in acetone forms a very efficient agent to remove silver chromate. Lead chromate substitution can be used with any material containing silver chromate stained structures. However, for better preservation of the non-impregnated background, aldehyde perfusion and impregnation according to the following technique are recommended.
Modified Golgi method for optimal ultrastructuralpreservation (1) The animals are anesthetized with pentobarbital and perfused with a mixture of glutaraldehyde (5 ~ ) and formaldehyde (4 ~o~) in 0.08 M cacodylate buffer at pH 7.3. Two-stage perfusion 6 can also give good results. (2) The brain is removed and stored overnight in 4 ~o formaldehyde in 0.08 M cacodylate buffer at pH 7.3.
Fig. 3. Election micrograph <'Jl"lhc main trunk of the peripheral mitral dend!it~ -hov, nil+ ~ i Open arrows point to synapli~: contacts. 31.500. Fig. 4. Secondary ramification of the s a m e dendrite s h o w n in fig. 2, in the interglomerulal ncu~,~ni!u 31,500. Figs. 5 a n d 6. Intraglomerular ramifications of the mitral dendrite s h o w n in k~g 2 31,50~!
343 (3) Three changes, two days each, in: potassium dichromate, l g; potassium chromate, 2 g; distilled water, 100 ml. (4) Two or 3 days in 3 ~o potassium dichromate with two daily changes. (5) Rinsing for a few seconds in distilled water and transfer to 1 o/~,silver nitrate for a minimum of 3 days. The silver solution should be changed at least twice before attempting lead chromate substitution. Lead chromate substitution (1) The fragments of impregnated material are sectioned with a razor blade, either by hand or by means of a tissue chopper (for example, the Sorvall TC2 microtome). The sections thus obtained can be kept almost indefinitely in 2 ~/o silver nitrate in the dark. (2) The sections, which are processed in 20 ml plastic vials, are transferred for 5 min to: 2 ~ silver nitrate, 1 vol. ; 2 ~o boric acid, 2 vol. (3) Three changes of 7 min each, at 20 °C in the following solution : 6 ~o lead lactate, l vol.; 2 ~o boric acid, 2 vol. (4) Two changes, 30 min each, in: 4~o barium lactate, 1 vol. : 2% silver nitrate, 1 vol. ; 2 ~o boric acid, 4 vol. (5) Two changes, 15 rain each, in 2 ~ silver nitrate. (6) Two changes, 5 min each, in 2 }/o potassium nitrate. (7) Overnight in : 4 ~ potassium dichromate, 3 vol. ; 4 ~o osmium tetroxide, I vol. (8) Acetones of ascending concentration (20 ~o, 40 °/o and 60 7/0, 10 rain each). (9) Three changes, 1 h each, in plastic vials, in the following solution : ammonium fluoride, l g; ammonium thiocyanate, 2 g; water, 40 ml; acetone, 60 ml. (10) Acetones of ascending concentrations (60~o, 80/0, o/ 100/o, o, 10 rain each) followed by embedding in epoxy resin, according to standard techniques. During all stages efficient shaking is recommended, to insure an even reaction. The results can be seen under incident light and, then, with electron microscopy. Under incident light (Figs. 1 and 2) the stained structures appear as yellow or greenyellow, depending on the degree of substitution. In general, cell bodies and dendrites appear much more yellow than axons and glial processes which show, as a rule, a greenish coloration. The electron photomicrographs (Figs. 3-6) show a remarkable degree of preservation of those structures which constitute the non-stained background of Golgistained material. The membranes of these non-impregnated structures are usually well outlined and organelles (mitochondria, endoplasmic reticulum, microtubules) are easily identifiable even though their fine details may be lost. Synapses are clearly seen, as well as their associated synaptic vesicles. Those structures that were originally impregnated by the Golgi method appear more or less filled with granules of lead chromate, often accumulated under the membrane. Inside these processes, mitochondria, microtubules and, sometimes, endoplasmic reticulum can be seen, although poorly preserved. The accumulation of the lead chromate granules under the membranes makes it difficult to identify synaptic contacts. Occasionally, however, these are evident (Figs. 3 and 4), whereas the techniques of direct observations of non-modified
344 silver c h r o m a t e seldom permit lhe identification of synapses on the impregnated p;~li~ les. As yet the present technique c a n n o t be used to visualize a l l t h e synapses prese~t o~ the surface of a given stained structure. However, some synapses can be demc)nst~tted and used to clarify some of the synaptic relationships of structures previously identified in Golgi-stained material. Supported by G r a n t MA4183 of the Medical Research Council of C a n a d a .
1 BLACKSTAD, T. W., Electron microscopy of Golgi preparations. In W. J. H. NAUTAAND S. O~ E. EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Heidelberg, 1970, pp. 186-216. 2 BLACKSTAD, T. W., Golgi preparations for electron microscopy: controlled reduction of the silver chromate by ultraviolet illumination. In M. SANTIM (Ed.), Proceedings Of the Golgi Centennial Symposium, Raven Press, New York, 1975, pp. 123-132. 3 BLACKSTAD, T. W., AND KJAERHEIM, A., Special axodendritic synapses in the hippocampal cortex. Electron and light microscopic studies on the layer of the mossy fibers, J. comp. Nem'ol., 117 (1961) 133-159. 4 CHAN-PALAY, V., AND PALAY, S. L., High voltage electron microscopy of rapid Golgi preparations, Z. Anat. Entwick/.-Gesch., 137 (1972) 125-152. 5 CHAN-PALAY, V., AND PALAY, S. L., The form ofvelate astrocytes in the cerebellar cortex of monkey and rat: high voltage electron microscopy of rapid Golgi preparations, 2". Anat. Entwickl.-Gesch., 138 (1972) 1-19. 6 PETERS,A., The fixation of central nervous tissue and the analysis of electron micrographs of the neuropil, with special reference to the cerebral cortex. In W. J. H. NAUTAAND S. O. E. EBaESSON (Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Heidelberg, 1970, pp. 56, 76. 7 RAMON-MOLINER, E., AND FERRARI, J., Electron microscopy of previously identified cells and processes within the central nervous system, J. Neurocytok, 1 (1972) 85-100. 8 STELE,W. K., Correlation of retinal cytoarchitecture and ultrastructure in Golgi preparations. Anat. Rec., 153 (1965) 389-397. 9 STELE,W. K., The structure and relationships of horizontal cells and photoreceptor bipolar synaptic complexes in goldfish retina, Amer. J. Anat., 121 (1967) 401 424.
339
Short Communications
Electron microscopy of golgi-stained material following substitution
lead chromate
E. RAMON-MOLINER AND J. FERRARI D~partement d'Anatomie, Facultd de Mddecine, Universitd de Sherbrooke, Sherbrooke, Que. (Canada)
(Accepted October 23rd, 1975)
The advantages of combining electron microscopy with the study of Golgistained material are now well documented. A significant advance was made by StellS,% Blackstad and Kjaerheim a, and Blackstad 1,2 who demonstrated that direct electron microscopy of previously Golgi-stained material was possible and that a surprising degree of preservation of ultrastructural detail could be achieved despite the chemical insults inflicted upon the tissue. However, direct electron microscopy of non-modified Golgi material has a number of disadvantages and difficulties1,7. For this reason, attempts were made to modify the silver chromate impregnation in order to render it less opaque to the electron beam and facilitate ultramicrotomy. Blackstad 1,~ proved that it was possible to obtain a partial 'de-impregnation' and demonstrated that the Golgi impregnation does not necessarily destroy ultrastructural details. Chan-Palay and Palay 4,a brought the innovation of studying Golgi-stained structures in sections several microns thick by means of high voltage electron microscopy. The present authors 7 have partially transformed the silver chromate precipitate present in Golgistained structures into lead chromate. When this partial replacement was followed by a treatment in a solution containing thiosulphate ions, only lead chromate was left in the tissue. This resulted in a depleted impregnation of the originally Golgi-stained structures without fading of their outlines. By using incident light to study the Araldite-embedded material prior to ultramicrotomy, stained structures appear as bright yellow against the dark background (Figs. I and 2). However, the degree of preservation of the fine ultrastructure was poor and the technique was, in general, difficult to control. The present report presents a method for controlling the degree of lead chromate substitution and improving ultrastructural preservation. One can distinguish the following stages in the performance of the technique: graded substitution, elimination of unwanted lead ions in the background, osmication, removal of unsubstituted silver chromate, dehydration, and embedding in an epoxy resin. In the substitution stage, the sections containing Golgi-stained structures are
340
Fig. I. Fragment of olfactory bulb of rat embedded in Araldite and showing under incident light mitral cells and periglomerular neurons impregnated with lead chromate, x 30. Fig. 2. Trimmed Araldite block showing under incident light the peripheral dendrite of a mitral celt (on the left). The secondary mitral dendrites of the external plexiform layer can be seen on the right. :-~ 120.
341 immersed in a solution of lead lactate buffered with boric acid. Of all the salts tried (nitrate, acetate, chlorate, chloride) lead lactate (K and K Laboratories, Plainview, N.Y. 11803) was the only one that permitted a graded reaction. Lead lactate does not give a massive and nearly instantaneous substitution. At room temperature, no replacement by lead chromate takes place before 10 rain, and at temperatures near 4 °C the reaction is considerably slowed. The presence of small amounts of silver nitrate acts as an inhibitor and for this reason it becomes necessary to change the solution of lead lactate to remove any traces of silver. The concentration of lead lactate does not seem to influence the rate of substitution. When buffered with boric acid, at pH 5, the reaction begins after 15 min. F r o m this moment on, it seems to take place at an accelerated rate so that after half an hour the amount of substitution is excessive. The addition of small amounts of silver nitrate can prolong the substitution time, but the reaction is irregular and difficult to control. With the p H higher than 5, the degree of substitution is lower but this is associated with background turbidity, due to lead precipitation. Once the desired degree of substitution is obtained, it is necessary to stop it without trapping lead in the background. In the absence of lactate ions, which appear to act as a chelating agent, lead is bound to the tissue in such a way that it will react at later stages with the chromate ions liberated by thiocyanate, giving a turbid background and a massive unwanted secondary substitution. Immersing the tissue in a solution containing silver, barium, and lactate ions, at pH 5, arrests the substitution process and washes away all the lead present in the background, without removing the lead chromate already formed within the stained structures. Osmication is carried out in the presence of dichromate ions to prevent excessive darkening of the tissue. Dichromate ions also appear to promote the formation of lead chromate in the stained structures in such a way that it will not diffuse and fade under the action of thiocyanate. The selective removal of non-replaced silver chromate occurs in an acetone solution of a m m o n i u m fluoride and a m m o n i u m thiocyanate. A m m o n i u m fluoride prevents the diffusion of lead chromate, since lead fluoride is highly insoluble in acetone. A m m o n i u m fluoride also can redissolve any metallic silver which may have formed in the background. A m m o n i u m thiocyanate dissolved in acetone forms a very efficient agent to remove silver chromate. Lead chromate substitution can be used with any material containing silver chromate stained structures. However, for better preservation of the non-impregnated background, aldehyde perfusion and impregnation according to the following technique are recommended.
Modified Golgi method for optimal ultrastructuralpreservation (1) The animals are anesthetized with pentobarbital and perfused with a mixture of glutaraldehyde (5 ~ ) and formaldehyde (4 ~o~) in 0.08 M cacodylate buffer at pH 7.3. Two-stage perfusion 6 can also give good results. (2) The brain is removed and stored overnight in 4 ~o formaldehyde in 0.08 M cacodylate buffer at pH 7.3.
Fig. 3. Election micrograph <'Jl"lhc main trunk of the peripheral mitral dend!it~ -hov, nil+ ~ i Open arrows point to synapli~: contacts. 31.500. Fig. 4. Secondary ramification of the s a m e dendrite s h o w n in fig. 2, in the interglomerulal ncu~,~ni!u 31,500. Figs. 5 a n d 6. Intraglomerular ramifications of the mitral dendrite s h o w n in k~g 2 31,50~!
343 (3) Three changes, two days each, in: potassium dichromate, l g; potassium chromate, 2 g; distilled water, 100 ml. (4) Two or 3 days in 3 ~o potassium dichromate with two daily changes. (5) Rinsing for a few seconds in distilled water and transfer to 1 o/~,silver nitrate for a minimum of 3 days. The silver solution should be changed at least twice before attempting lead chromate substitution. Lead chromate substitution (1) The fragments of impregnated material are sectioned with a razor blade, either by hand or by means of a tissue chopper (for example, the Sorvall TC2 microtome). The sections thus obtained can be kept almost indefinitely in 2 ~/o silver nitrate in the dark. (2) The sections, which are processed in 20 ml plastic vials, are transferred for 5 min to: 2 ~ silver nitrate, 1 vol. ; 2 ~o boric acid, 2 vol. (3) Three changes of 7 min each, at 20 °C in the following solution : 6 ~o lead lactate, l vol.; 2 ~o boric acid, 2 vol. (4) Two changes, 30 min each, in: 4~o barium lactate, 1 vol. : 2% silver nitrate, 1 vol. ; 2 ~o boric acid, 4 vol. (5) Two changes, 15 rain each, in 2 ~ silver nitrate. (6) Two changes, 5 min each, in 2 }/o potassium nitrate. (7) Overnight in : 4 ~ potassium dichromate, 3 vol. ; 4 ~o osmium tetroxide, I vol. (8) Acetones of ascending concentration (20 ~o, 40 °/o and 60 7/0, 10 rain each). (9) Three changes, 1 h each, in plastic vials, in the following solution : ammonium fluoride, l g; ammonium thiocyanate, 2 g; water, 40 ml; acetone, 60 ml. (10) Acetones of ascending concentrations (60~o, 80/0, o/ 100/o, o, 10 rain each) followed by embedding in epoxy resin, according to standard techniques. During all stages efficient shaking is recommended, to insure an even reaction. The results can be seen under incident light and, then, with electron microscopy. Under incident light (Figs. 1 and 2) the stained structures appear as yellow or greenyellow, depending on the degree of substitution. In general, cell bodies and dendrites appear much more yellow than axons and glial processes which show, as a rule, a greenish coloration. The electron photomicrographs (Figs. 3-6) show a remarkable degree of preservation of those structures which constitute the non-stained background of Golgistained material. The membranes of these non-impregnated structures are usually well outlined and organelles (mitochondria, endoplasmic reticulum, microtubules) are easily identifiable even though their fine details may be lost. Synapses are clearly seen, as well as their associated synaptic vesicles. Those structures that were originally impregnated by the Golgi method appear more or less filled with granules of lead chromate, often accumulated under the membrane. Inside these processes, mitochondria, microtubules and, sometimes, endoplasmic reticulum can be seen, although poorly preserved. The accumulation of the lead chromate granules under the membranes makes it difficult to identify synaptic contacts. Occasionally, however, these are evident (Figs. 3 and 4), whereas the techniques of direct observations of non-modified
344 silver c h r o m a t e seldom permit lhe identification of synapses on the impregnated p;~li~ les. As yet the present technique c a n n o t be used to visualize a l l t h e synapses prese~t o~ the surface of a given stained structure. However, some synapses can be demc)nst~tted and used to clarify some of the synaptic relationships of structures previously identified in Golgi-stained material. Supported by G r a n t MA4183 of the Medical Research Council of C a n a d a .
1 BLACKSTAD, T. W., Electron microscopy of Golgi preparations. In W. J. H. NAUTAAND S. O~ E. EBBESSON(Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Heidelberg, 1970, pp. 186-216. 2 BLACKSTAD, T. W., Golgi preparations for electron microscopy: controlled reduction of the silver chromate by ultraviolet illumination. In M. SANTIM (Ed.), Proceedings Of the Golgi Centennial Symposium, Raven Press, New York, 1975, pp. 123-132. 3 BLACKSTAD, T. W., AND KJAERHEIM, A., Special axodendritic synapses in the hippocampal cortex. Electron and light microscopic studies on the layer of the mossy fibers, J. comp. Nem'ol., 117 (1961) 133-159. 4 CHAN-PALAY, V., AND PALAY, S. L., High voltage electron microscopy of rapid Golgi preparations, Z. Anat. Entwick/.-Gesch., 137 (1972) 125-152. 5 CHAN-PALAY, V., AND PALAY, S. L., The form ofvelate astrocytes in the cerebellar cortex of monkey and rat: high voltage electron microscopy of rapid Golgi preparations, 2". Anat. Entwickl.-Gesch., 138 (1972) 1-19. 6 PETERS,A., The fixation of central nervous tissue and the analysis of electron micrographs of the neuropil, with special reference to the cerebral cortex. In W. J. H. NAUTAAND S. O. E. EBaESSON (Eds.), Contemporary Research Methods in Neuroanatomy, Springer, Heidelberg, 1970, pp. 56, 76. 7 RAMON-MOLINER, E., AND FERRARI, J., Electron microscopy of previously identified cells and processes within the central nervous system, J. Neurocytok, 1 (1972) 85-100. 8 STELE,W. K., Correlation of retinal cytoarchitecture and ultrastructure in Golgi preparations. Anat. Rec., 153 (1965) 389-397. 9 STELE,W. K., The structure and relationships of horizontal cells and photoreceptor bipolar synaptic complexes in goldfish retina, Amer. J. Anat., 121 (1967) 401 424.