Aeta Neuropathol (1992) 84: 198- 201
HeuAC wathobgka ~) Springer-Verlag1992
Short original communications Microwave-enhanced silver staining of degenerating neuronal processes B. Van Deuren, J. Van Reempts, and M. Borgers Laboratory of Neuropathology, Department of Morphology, Janssen Research Foundation, B-2340 Beerse, Belgium Received December 27, 1991/Revised, accepted February 17, 1992
Summary. A simple and rapid m e t h o d for light and electron microscopic visualization of degenerating neuronal processes and axon terminals is described. H u n d r e d - m i c r o m e t e r v i b r a t o m e sections of perfusionfixed rat brain were incubated briefly in a 5 % silver nitrate solution in a conventional microwave oven. A f t e r a rinse in 1 % acetic acid, the sections were silver enhanced. Differentiation and counterstaining was done r e s p e c t i v e l y in ethanol 100 % and cresyl violet. In the light microscope, degenerating neuronal processes a p p e a r e d as black dots against a clear background. A r e a s of calcification were also positively stained. T h e presence of silver deposits in degenerating presynaptic terminals and dendrites was confirmed ultrastructurally.
p e n e t r a t i o n and diffusion. We therefore a d a p t e d the m e t h o d of Grimelius [6]. T h e use of high concentrations of silver and a commercially available enhancer in combination with microwave exposure resulted in a fast, reliable technique for the staining of degenerating neuronal processes, including axon terminals. T h e technique is perfectly suitable for thick v i b r a t o m e sections which can be w h o l e - m o u n t e d as survey sections or routinely e m b e d d e d in E p o n for detailed light and electron microscopy.
Key words: Brain - Silver staining - Microwave technique - A x o n terminal - Histology
Unilateral hypoxia-ischemia was induced in infant rats by a combination of carotid artery ligation and low 02 exposure [7]. Excitotoxic lesions of the limbic system were induced in rats after i.v. injection of kainate [2]. Focal cerebral infarcts were photochemically produced in the sensorimotor cortex of rats after i.v. injection of rose bengal and transcranial illumination of the brain [15]. Global incomplete ischemia was induced in rats by a combination of bilateral carotid artery occlusion and severe hypotension [14].
M e t h o d s which e m p l o y silver i m p r e g n a t i o n of neurons and their processes are widely used in neuropathology. T h e procedures are usually time consuming and the staining m e c h a n i s m is not always clearly understood. T h e majority of these m e t h o d s have b e e n d e v e l o p e d for paraffin sections. Our interest was to stain degenerating axon endings in 100-~m v i b r a t o m e sections of whole rat brain. T h e available m e t h o d s , such as F i n k - H e i m e r ' s , Bielschowsky's, Grimelius' silver stains and their m a n y modifications [3, 4, 6, 11, 12], apart f r o m being time consuming, resulted in overstaining in thick sections rapidly. In addition, due to the long incubation periods, sections easily t e n d e d to b e c o m e detached f r o m the slides. T h e interest in the use of microwave ovens in histology laboratories is continuously growing [1, 9]. Microwave techniques offer the possibility of considerably reducing staining time and improving reagent
Correspondence to: B. Van Deuren (address see above)
Materials and methods Induction of neuronal degeneration
Fixation and staining Fixation was initiated by perfusion with a mixture of 2% paraformaldehyde and 2.5 % glutaraldehyde in a 0.1 M phosphate buffer. After perfusion, the brains remained in the skull in the same fixative for at least I day. Coronal 100%tmvibratome sections were prepared in the lesioned area as well as in retro- and anterograde sites. They were mounted on gelatine-coated slides. Staining was carried out after drying at 37~ for 2-4 days. A conventional microwave oven (Miele, Electronic M696) was used. The sections were stained in 200 ml of a 5 % silver nitrate solution in distilled water.The jar was placed in the oven for 40 s at 700 Watt, to reach a temperature of 45 ~ was immediately followed by a staining period of 5 rain at 150 Watt. The final temperature amounted to 70~ After microwave incubation, sections were rinsed in 1% acetic acid for 1 rain under gentle shaking. Sections were then rinsed twice in distilled water and subjected to silver enhancement at room temperature for 2 to 6 rain (Intense BL kit,
199 Amersham). Counterstaining was carried out either with cresyl violet after 2 h differentiation in ethanol 100 % or with azureeosin. Finally the sections were routinely dehydrated, cleared and mounted with pertex. Control vibratome sections were stained with silver nitrate or with silver enhancer only. Staining of vibratome sections was compared with that of routinely embedded paraffin sections.
Electron microscopy For electron microscopy, intermediate 200-~tmvibratome sections were used. These sections were stained free-floating, using the same solutions and time intervals as described above. After staining, the samples were postfixed in osmium tetroxide, dehydrated in graded ethanol-series and embedded in Epon LX.
Results In the fight microscope, a positive staining reaction was characterized by the presence of numerous black dots which were clearly differentiated from unstained gray matter and light brown white m a t t e r (Fig. la). Size and localization of the silver deposits were indicative for axon endings. A t higher magnifications, it was obvious that the silver was present as separated dots which often coincided with the course of cell processes or covered entire neuronal cell bodies (Fig. lb). Localization and density of the silver precipitates d e p e n d e d upon the type of insult. Unilateral hypoxia-ischemia resulted in intense staining of hippocampus and cortex in the ligated hemisphere only.Thalamic nuclei were occasionally stained. Staining was absent in regions of reactive gliosis which developed after prolonged survival periods (Fig. la). Kainate administration resulted in a positive staining of specific hippocampal subfields and in amygdala. A f t e r survival times of 1-2 weeks, certain thalamic nuclei also became positive. A f t e r 5 weeks survival, the staining reaction became visible in the forebrain whereas in the hippocampus, silver deposits disappeared. Stroke lesions in the sensorimotor cortex resulted in retrograde staining of ventrolateral and posterior thalamic nuclei after 3 weeks. A r o u n d the infarct, a small n u m b e r of heavily stained neurons was found. The same neurons showed a positive reaction with Alizarine red, which is indicative for their high calcium content. Large black globules were also detected in the thalamus: they were earlier identified as calcareous concretions [16]. Shorter survival times occasionally resulted in silver deposits in the corpus callosum underlying the infarct. Global incomplete ischemia did not result in any positive staining reaction in selectively vulnerable areas after 1 week survival. In the electron microscope it was confirmed that the black deposits were largely confined to degenerating presynaptic terminals. Silver precipitates seemed to be located inside synaptic vesicles (Fig. 2). However, a considerable n u m b e r of dendritic processes also showed positive reaction product either localized inside the cytosol or inside mitochondria.
Fig. 1. a Coronal 100-gm vibratome section of rat brain, 1 week after unilateral carotid artery occlusion and hypoxic exposure. Microwave-enhanced silver staining clearly differentiates necrotic areas (black) from normal tissue. Damage is found in parietal cortex (C), hippocampus (H) and thalamus (T). Silver staining is absent in regions of reactive gliosis (arrow).White matter appears brown (arrowhead). Azure-eosin counterstaining, b Light microscopic detail of hypoxic hippocampal region from a. Degenerating axon terminals and dendritic processes become visible as black granular silver deposits (arrows) whereas neuronal cell bodies do not react (arrowhead)
Staining with the silver enhancer alone did not produce any reaction. When microwave incubation in silver nitrate was applied only, staining was barely visible. Omitting counterstaining and alcohol differentiation, resulted in a higher background. Paraffin sections could not be stained with this technique.
Discussion With the present technique, a clear, reproducible and homogeneous staining of degenerating neuronal processes could be o b t a i n e d . T h e use of a microwave oven led to considerable time sparing and a low background staining. This clear differentiation from the background makes it possible to detect subtle damage. Moreover, the high contrast offers the possibility of using the sections for automatic density measurements. The m e t h o d can be used on batches of slides and is very well
200
Fig. 2. Electron microscopicpicture of hypoxicrat brain cortex. Microwave-enhancedsilver staining reveals abundant positivelystained profiles (arrows) part of which are characteristic for axon terminals (inset)
suited to thick vibratome sections. However, a constant volume of 200 ml silver nitrate should be used during microwave incubation, since variations in the volume may result in different temperatures and energy absorption, and hence in variable staining results. The use of sections, dried for 2 to 4 days at 37 ~ is recommended. Longer drying periods give a weaker and less complete staining, whereas incomplete drying results in a higher background. The reason behind this difference is unknown. The simple silver enhancement with a commercially available kit greatly improves the visibility of subtle damage. This is due to enlargement of the silver granules which are deposited in the tissue during microwave incubation [10]. Further standardization and improvement of the technique can be expected when an industrial microwave oven is used. Ovens, especially designed for laboratory practice offer the possibility of exact temperature and time control and are equipped with a stirring device. For safety reasons, we were not able to use organic solvents in our microwave oven. Since in industrial ovens, the use of solvents is allowed, it might be possible to reduce also the time needed for differentiation in absolute ethanol.
The mechanism of the present staining technique is not completely clear [5, 13]. Probably the degeneration of axon endings may result in the formation of chemical groups, which are able to reduce silver ions. These active groups may be formed from disintegration products specific for axon endings. Silver deposits in dendritic processes and mitochondria might be explained by the presence of high amounts of intracellular calcium in degenerating cells. The reduction of silver ions is greatly enhanced by the energy supplied by the microwaves. Incubation up to 48 h (without microwave treatment) did not result in visible silver deposits. The time needed for incubation without microwaves is, thus, extremely long, suggesting that external energy must be supplied for the reduction of silver ions. Also, the penetration of the silver ions in the tissue is facilitated by the microwaves. During the silver enhancement, silver ions in the enhancer are in turn reduced by the earlier formed silver deposits in the tissue, resulting in microscopically visible silver granules. There is no clear explanation for the absence of silver staining after global cerebral ischemia. Damage in this model is restricted to pyramidal cell bodies in the CA1 subfield [14]. Moreover, no reduction of axon terminals
201 could be d e t e c t e d in this area, even n o t after p r o l o n g e d survival periods [8]. T h e r e f o r e it is very unlikely that p r e s y n a p t i c a x o n endings b e c o m e stained with this t e c h n i q u e in this m o d e l . Staining of d e g e n e r a t i n g a x o n endings m a y be very useful in e x p e r i m e n t a l neurology, for studying n e u r o n a l p a t h w a y s as well as p a t h o p h y s i o l o g y and t h e r a p y of brain disease. Using this t e c h n i q u e we w e r e able to show r e t r o g r a d e d e g e n e r a t i o n after cortical stroke. Also after chemical lesioning with kainate, t h e initial d a m a g e in a m y g d a l a and h i p p o c a m p u s was f o u n d to e x t e n d to o t h e r areas after l o n g e r survival times. T h e excellent differentiation using this m e t h o d m a y offer the possibility of easily q u a n t i f y i n g the d e g r e e of p r o t e c t i o n o b t a i n e d with c e r e b r o p r o t e c t i v e drugs. T h e present staining t e c h n i q u e can, t h e r e f o r e , be r e c o m m e n d e d as a helpful t o o l in the n e u r o p a t h o l o g y laboratory.
Acknowledgements. The authors are grateful to Mr. L. Leijssen and Mr. G. Jacobs for preparing the illustrations, and to Mrs. I. Gevers for typing the manuscript.
References 1. Brinn NT (1983) Rapid metallic histological staining using the microwave oven. J Histotechnol 6:125-129 2. Cronin J, Dudek FE (1988) Chronic seizures and collateral sprouting of dentate mossy fibers after kainic acid treatment in rats. Brain Res 474:181-184 3. Fernandez Pascual JS (1976) A new method for easy demonstration of argyrophill cells. Stain Technol 51:231-235 4. Fink RR Heimer L (1967) Two methods for selective impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res 4:369-374
5. Gallyas E Zaborszky l,Wolff JR (1980) Experimental studies of mechanisms involved in methods demonstrating axonal and terminal degeneration. Stain Technol 55:281-290 6. Grimelius L (1968) A silver nitrate stain for ct2-cells in human pancreatic islets. Acta Soc Med Upsal 73:243-270 7. Gunn AJ, Mydlar T, Bennet R, Faull RLM, Gorter S, Cook C, Johnston BM, Gluckman PD (1989) The neuroprotective actions of a calcium channel antagonist, flunarizine, in the infant rat. Pediatr Res 25:573-576 8. Kirino T, Tamura A, Sano K (1989) Persistent presynaptic terminals in the hippocampal CA1 sector following ischemic neuronal loss. J Cereb Blood Flow Metab 9:$554 9. Kok LE Boon ME (1990) Microwaves for microscopy. J Microsc 158:291-322 10. Lah JJ, Hayes DM, Burry RW (1990) A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry. J Histochem Cytochem 38:503-508 11. Lillie RD, Fullmer HM (1976) Histopathologic technic and practical histochemistry, 4th edn. McGraw Hill, New York, pp 682-692 12. Mallory FB (1961) Pathological technique. Hafner Publishing, New York, pp 158-160 13. Samuel EP (1953) The mechanism of silver staining. J Anat 87:278-287 14. Smith M-L, Bendek G, Dahlgren N, Ros6n I,WielochT, Siesj6 BK (1984) Models for studying long-term recovery following forebrain ischemia in the rat. II. A 2-vessel occlusion model. Acta Neurol Scand 69:385-401 15. Van Reempts J, Van Deuren B, Van de Ven M, Cornelissen F, Borgers M (1987) Flunarizine reduces cerebral infarct size after photochemically induced thrombosis in spontaneously hypertensive rats. Stroke 18:1113-1119 16. Van Reempts J,Van Deuren B, Borgers M, De Nollin S (1989) Calcification and glial reactivity in rat thalamus after photochemical neocortical stroke. J Cereb Blood Flow Metab 9 [Suppl 1]:$168
HeuAC wathobgka ~) Springer-Verlag1992
Short original communications Microwave-enhanced silver staining of degenerating neuronal processes B. Van Deuren, J. Van Reempts, and M. Borgers Laboratory of Neuropathology, Department of Morphology, Janssen Research Foundation, B-2340 Beerse, Belgium Received December 27, 1991/Revised, accepted February 17, 1992
Summary. A simple and rapid m e t h o d for light and electron microscopic visualization of degenerating neuronal processes and axon terminals is described. H u n d r e d - m i c r o m e t e r v i b r a t o m e sections of perfusionfixed rat brain were incubated briefly in a 5 % silver nitrate solution in a conventional microwave oven. A f t e r a rinse in 1 % acetic acid, the sections were silver enhanced. Differentiation and counterstaining was done r e s p e c t i v e l y in ethanol 100 % and cresyl violet. In the light microscope, degenerating neuronal processes a p p e a r e d as black dots against a clear background. A r e a s of calcification were also positively stained. T h e presence of silver deposits in degenerating presynaptic terminals and dendrites was confirmed ultrastructurally.
p e n e t r a t i o n and diffusion. We therefore a d a p t e d the m e t h o d of Grimelius [6]. T h e use of high concentrations of silver and a commercially available enhancer in combination with microwave exposure resulted in a fast, reliable technique for the staining of degenerating neuronal processes, including axon terminals. T h e technique is perfectly suitable for thick v i b r a t o m e sections which can be w h o l e - m o u n t e d as survey sections or routinely e m b e d d e d in E p o n for detailed light and electron microscopy.
Key words: Brain - Silver staining - Microwave technique - A x o n terminal - Histology
Unilateral hypoxia-ischemia was induced in infant rats by a combination of carotid artery ligation and low 02 exposure [7]. Excitotoxic lesions of the limbic system were induced in rats after i.v. injection of kainate [2]. Focal cerebral infarcts were photochemically produced in the sensorimotor cortex of rats after i.v. injection of rose bengal and transcranial illumination of the brain [15]. Global incomplete ischemia was induced in rats by a combination of bilateral carotid artery occlusion and severe hypotension [14].
M e t h o d s which e m p l o y silver i m p r e g n a t i o n of neurons and their processes are widely used in neuropathology. T h e procedures are usually time consuming and the staining m e c h a n i s m is not always clearly understood. T h e majority of these m e t h o d s have b e e n d e v e l o p e d for paraffin sections. Our interest was to stain degenerating axon endings in 100-~m v i b r a t o m e sections of whole rat brain. T h e available m e t h o d s , such as F i n k - H e i m e r ' s , Bielschowsky's, Grimelius' silver stains and their m a n y modifications [3, 4, 6, 11, 12], apart f r o m being time consuming, resulted in overstaining in thick sections rapidly. In addition, due to the long incubation periods, sections easily t e n d e d to b e c o m e detached f r o m the slides. T h e interest in the use of microwave ovens in histology laboratories is continuously growing [1, 9]. Microwave techniques offer the possibility of considerably reducing staining time and improving reagent
Correspondence to: B. Van Deuren (address see above)
Materials and methods Induction of neuronal degeneration
Fixation and staining Fixation was initiated by perfusion with a mixture of 2% paraformaldehyde and 2.5 % glutaraldehyde in a 0.1 M phosphate buffer. After perfusion, the brains remained in the skull in the same fixative for at least I day. Coronal 100%tmvibratome sections were prepared in the lesioned area as well as in retro- and anterograde sites. They were mounted on gelatine-coated slides. Staining was carried out after drying at 37~ for 2-4 days. A conventional microwave oven (Miele, Electronic M696) was used. The sections were stained in 200 ml of a 5 % silver nitrate solution in distilled water.The jar was placed in the oven for 40 s at 700 Watt, to reach a temperature of 45 ~ was immediately followed by a staining period of 5 rain at 150 Watt. The final temperature amounted to 70~ After microwave incubation, sections were rinsed in 1% acetic acid for 1 rain under gentle shaking. Sections were then rinsed twice in distilled water and subjected to silver enhancement at room temperature for 2 to 6 rain (Intense BL kit,
199 Amersham). Counterstaining was carried out either with cresyl violet after 2 h differentiation in ethanol 100 % or with azureeosin. Finally the sections were routinely dehydrated, cleared and mounted with pertex. Control vibratome sections were stained with silver nitrate or with silver enhancer only. Staining of vibratome sections was compared with that of routinely embedded paraffin sections.
Electron microscopy For electron microscopy, intermediate 200-~tmvibratome sections were used. These sections were stained free-floating, using the same solutions and time intervals as described above. After staining, the samples were postfixed in osmium tetroxide, dehydrated in graded ethanol-series and embedded in Epon LX.
Results In the fight microscope, a positive staining reaction was characterized by the presence of numerous black dots which were clearly differentiated from unstained gray matter and light brown white m a t t e r (Fig. la). Size and localization of the silver deposits were indicative for axon endings. A t higher magnifications, it was obvious that the silver was present as separated dots which often coincided with the course of cell processes or covered entire neuronal cell bodies (Fig. lb). Localization and density of the silver precipitates d e p e n d e d upon the type of insult. Unilateral hypoxia-ischemia resulted in intense staining of hippocampus and cortex in the ligated hemisphere only.Thalamic nuclei were occasionally stained. Staining was absent in regions of reactive gliosis which developed after prolonged survival periods (Fig. la). Kainate administration resulted in a positive staining of specific hippocampal subfields and in amygdala. A f t e r survival times of 1-2 weeks, certain thalamic nuclei also became positive. A f t e r 5 weeks survival, the staining reaction became visible in the forebrain whereas in the hippocampus, silver deposits disappeared. Stroke lesions in the sensorimotor cortex resulted in retrograde staining of ventrolateral and posterior thalamic nuclei after 3 weeks. A r o u n d the infarct, a small n u m b e r of heavily stained neurons was found. The same neurons showed a positive reaction with Alizarine red, which is indicative for their high calcium content. Large black globules were also detected in the thalamus: they were earlier identified as calcareous concretions [16]. Shorter survival times occasionally resulted in silver deposits in the corpus callosum underlying the infarct. Global incomplete ischemia did not result in any positive staining reaction in selectively vulnerable areas after 1 week survival. In the electron microscope it was confirmed that the black deposits were largely confined to degenerating presynaptic terminals. Silver precipitates seemed to be located inside synaptic vesicles (Fig. 2). However, a considerable n u m b e r of dendritic processes also showed positive reaction product either localized inside the cytosol or inside mitochondria.
Fig. 1. a Coronal 100-gm vibratome section of rat brain, 1 week after unilateral carotid artery occlusion and hypoxic exposure. Microwave-enhanced silver staining clearly differentiates necrotic areas (black) from normal tissue. Damage is found in parietal cortex (C), hippocampus (H) and thalamus (T). Silver staining is absent in regions of reactive gliosis (arrow).White matter appears brown (arrowhead). Azure-eosin counterstaining, b Light microscopic detail of hypoxic hippocampal region from a. Degenerating axon terminals and dendritic processes become visible as black granular silver deposits (arrows) whereas neuronal cell bodies do not react (arrowhead)
Staining with the silver enhancer alone did not produce any reaction. When microwave incubation in silver nitrate was applied only, staining was barely visible. Omitting counterstaining and alcohol differentiation, resulted in a higher background. Paraffin sections could not be stained with this technique.
Discussion With the present technique, a clear, reproducible and homogeneous staining of degenerating neuronal processes could be o b t a i n e d . T h e use of a microwave oven led to considerable time sparing and a low background staining. This clear differentiation from the background makes it possible to detect subtle damage. Moreover, the high contrast offers the possibility of using the sections for automatic density measurements. The m e t h o d can be used on batches of slides and is very well
200
Fig. 2. Electron microscopicpicture of hypoxicrat brain cortex. Microwave-enhancedsilver staining reveals abundant positivelystained profiles (arrows) part of which are characteristic for axon terminals (inset)
suited to thick vibratome sections. However, a constant volume of 200 ml silver nitrate should be used during microwave incubation, since variations in the volume may result in different temperatures and energy absorption, and hence in variable staining results. The use of sections, dried for 2 to 4 days at 37 ~ is recommended. Longer drying periods give a weaker and less complete staining, whereas incomplete drying results in a higher background. The reason behind this difference is unknown. The simple silver enhancement with a commercially available kit greatly improves the visibility of subtle damage. This is due to enlargement of the silver granules which are deposited in the tissue during microwave incubation [10]. Further standardization and improvement of the technique can be expected when an industrial microwave oven is used. Ovens, especially designed for laboratory practice offer the possibility of exact temperature and time control and are equipped with a stirring device. For safety reasons, we were not able to use organic solvents in our microwave oven. Since in industrial ovens, the use of solvents is allowed, it might be possible to reduce also the time needed for differentiation in absolute ethanol.
The mechanism of the present staining technique is not completely clear [5, 13]. Probably the degeneration of axon endings may result in the formation of chemical groups, which are able to reduce silver ions. These active groups may be formed from disintegration products specific for axon endings. Silver deposits in dendritic processes and mitochondria might be explained by the presence of high amounts of intracellular calcium in degenerating cells. The reduction of silver ions is greatly enhanced by the energy supplied by the microwaves. Incubation up to 48 h (without microwave treatment) did not result in visible silver deposits. The time needed for incubation without microwaves is, thus, extremely long, suggesting that external energy must be supplied for the reduction of silver ions. Also, the penetration of the silver ions in the tissue is facilitated by the microwaves. During the silver enhancement, silver ions in the enhancer are in turn reduced by the earlier formed silver deposits in the tissue, resulting in microscopically visible silver granules. There is no clear explanation for the absence of silver staining after global cerebral ischemia. Damage in this model is restricted to pyramidal cell bodies in the CA1 subfield [14]. Moreover, no reduction of axon terminals
201 could be d e t e c t e d in this area, even n o t after p r o l o n g e d survival periods [8]. T h e r e f o r e it is very unlikely that p r e s y n a p t i c a x o n endings b e c o m e stained with this t e c h n i q u e in this m o d e l . Staining of d e g e n e r a t i n g a x o n endings m a y be very useful in e x p e r i m e n t a l neurology, for studying n e u r o n a l p a t h w a y s as well as p a t h o p h y s i o l o g y and t h e r a p y of brain disease. Using this t e c h n i q u e we w e r e able to show r e t r o g r a d e d e g e n e r a t i o n after cortical stroke. Also after chemical lesioning with kainate, t h e initial d a m a g e in a m y g d a l a and h i p p o c a m p u s was f o u n d to e x t e n d to o t h e r areas after l o n g e r survival times. T h e excellent differentiation using this m e t h o d m a y offer the possibility of easily q u a n t i f y i n g the d e g r e e of p r o t e c t i o n o b t a i n e d with c e r e b r o p r o t e c t i v e drugs. T h e present staining t e c h n i q u e can, t h e r e f o r e , be r e c o m m e n d e d as a helpful t o o l in the n e u r o p a t h o l o g y laboratory.
Acknowledgements. The authors are grateful to Mr. L. Leijssen and Mr. G. Jacobs for preparing the illustrations, and to Mrs. I. Gevers for typing the manuscript.
References 1. Brinn NT (1983) Rapid metallic histological staining using the microwave oven. J Histotechnol 6:125-129 2. Cronin J, Dudek FE (1988) Chronic seizures and collateral sprouting of dentate mossy fibers after kainic acid treatment in rats. Brain Res 474:181-184 3. Fernandez Pascual JS (1976) A new method for easy demonstration of argyrophill cells. Stain Technol 51:231-235 4. Fink RR Heimer L (1967) Two methods for selective impregnation of degenerating axons and their synaptic endings in the central nervous system. Brain Res 4:369-374
5. Gallyas E Zaborszky l,Wolff JR (1980) Experimental studies of mechanisms involved in methods demonstrating axonal and terminal degeneration. Stain Technol 55:281-290 6. Grimelius L (1968) A silver nitrate stain for ct2-cells in human pancreatic islets. Acta Soc Med Upsal 73:243-270 7. Gunn AJ, Mydlar T, Bennet R, Faull RLM, Gorter S, Cook C, Johnston BM, Gluckman PD (1989) The neuroprotective actions of a calcium channel antagonist, flunarizine, in the infant rat. Pediatr Res 25:573-576 8. Kirino T, Tamura A, Sano K (1989) Persistent presynaptic terminals in the hippocampal CA1 sector following ischemic neuronal loss. J Cereb Blood Flow Metab 9:$554 9. Kok LE Boon ME (1990) Microwaves for microscopy. J Microsc 158:291-322 10. Lah JJ, Hayes DM, Burry RW (1990) A neutral pH silver development method for the visualization of 1-nanometer gold particles in pre-embedding electron microscopic immunocytochemistry. J Histochem Cytochem 38:503-508 11. Lillie RD, Fullmer HM (1976) Histopathologic technic and practical histochemistry, 4th edn. McGraw Hill, New York, pp 682-692 12. Mallory FB (1961) Pathological technique. Hafner Publishing, New York, pp 158-160 13. Samuel EP (1953) The mechanism of silver staining. J Anat 87:278-287 14. Smith M-L, Bendek G, Dahlgren N, Ros6n I,WielochT, Siesj6 BK (1984) Models for studying long-term recovery following forebrain ischemia in the rat. II. A 2-vessel occlusion model. Acta Neurol Scand 69:385-401 15. Van Reempts J, Van Deuren B, Van de Ven M, Cornelissen F, Borgers M (1987) Flunarizine reduces cerebral infarct size after photochemically induced thrombosis in spontaneously hypertensive rats. Stroke 18:1113-1119 16. Van Reempts J,Van Deuren B, Borgers M, De Nollin S (1989) Calcification and glial reactivity in rat thalamus after photochemical neocortical stroke. J Cereb Blood Flow Metab 9 [Suppl 1]:$168
