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Silver Staining of Senile Plaques and Neurofibrillary Tangles in Paraffin Sections
Path. Res. Pract. 187, 1045-1049 (1991)
Rapid Communication
Silver Staining of Senile Plaques and Neurofibrillary Tangles in Paraffin Sections A Simple and Effective Method E. Reusche Institute of Pathology, Medical University Lubeck, FRG
SUMMARY
A one-step silver staining method using a protective colloidal developer of gelatin and formic acid was originally established for demonstration of argyrophilic nucleolus organizer regions by controlled reduction of silver. We describe here a new application of this silver technique that can easily be performed to demonstrate senile plaques (SP) and neurofibrillary tangles (NFT) on paraffin sections. Preliminary results in ten cases of senile dementia of Alzheimer type and five cases of amyloid congophilic angiopathy showed a reliable demonstration of amyloid and neuritic type SP as well as NFT in all 15 cases. In addition, deposits of perivascular amyloid and areas of fibrillar amyloid, the diffuse type of senile plaques, were revealed. The success seems to depend particularly on the low concentration of formic acid in the developer, which might be the responsible agent for the careful revealing of buried argyrophilic structures in SP and NFT. The staining features were quite similar to those with anti-~ and anti-tau immunostaining. This result suggests a high specificity of this method for extracellular and intraneuronal cerebral amyloid. The detailed staining of delicate morphological structures points to a high sensitivity for cerebral amyloids, senile plaques and neurofibrillary tangles, respectively, by this simple and inexpensive method.
Introduction Since Alzheimer's first description in 19061 of a dementia in a 57-year-old female the characteristic lesions of senile plaques and neurofibrillary tangles have been demonstrated by complicated silver impregnation. The methods of Bielschowsky and von Braunmiihl4 are used on small frozen sections of formalin-fixed material, Bodian's5 and a modified Palmgren's method by Cross ll and are applied on paraffin sections. A one-step silver staining method was originally established for demonstration of argyrophilic nucleolus organizer regions (NOR) on the optical level by the aid of a protective colloidal developer of gelatin and formic acid 16. Recently this sensitive method with a controlled reduction © 1991 by Gustav Fischer Verlag, Stuttgart
of silver has come into use for counting in tumour pathology NOR's in size and number as a possible marker for proliferation or malignancy12,22. While investigating brain tumours by this silver technique, several indistinct, faintly stained senile plaques were found coincidentally in a small tumorous cortical area of a 65-year-old male prompting a careful evaluation of this method.
Material and Methods Between 1985 and 1990 we selected 10 patients with the clinical and/or morphological diagnosis of senile dementia of Alzheimer type (SDAT), 67-88 years of age and brain weights of 0344-0338/9110187 -1 045 $3.50/0
1046 . E. Reusche 950-1120 g; 5 patients with an amyloid congophilic angiopathy (ACA), age 65-87 years; 4 young patients and a 72-year-old
female served as a negative control group; in 5 patients we compared our silver technique with v. Braunmiihl's silver impregnation. Three IL thick sections from different regions (frontal cortex, hippocampus, amygdala and occipital cortex) were stained with Nissl, HE, PAS and congo red stains. The silver staining procedure was performed with slight modifications according to the method of Howell and Black l6 : 1) The colloidal developer was prepared by mixing 1 volume, 2 % gelatin, which had been dissolved in 1 % formic acid (stable for maximum 2 weeks), with 2 volumes of 50 % silver nitrate solution (maturing for several weeks). 2) Deparaffinized sections, 10 IL thick, were incubated with the developer-silver solution in a moist dark chamber, in horizontal position and at room temperature for 60-120 minutes. During the incubation they were covered with a coverslip. Repeated rinsing with distilled water followed. 3) To reduce the brown colour of the background, the slides were subsequently given into 0,1 % gold chloride for 2 minutes, again followed by repeated rinsing. 4) After 2 minutes of incubation in a 3 % sodium-thiosulfate solution the slides were rinsed for 30 minutes in tap water. 5) Finally the sections were dehydrated, cleared and mounted with a synthetic resin (Eukitt, Kindler).
Fig. 1. Low power micrographs of corresponding temporal cortical areas with numerous SP: a) silver (inset: "classical" plaque, x 400); b) anti-beta protein.
Table 1. Counts of senile plaques and neurofibrillary tangles: comparison of silver staining and immuno-reaction in corresponding cortical fields of 8 mm 2 each in five topographically different specimens Senile plaques ~-protein silver
Neurofibrillary tangles silver tau-protein
218 174
202 161
160
52
70 56
80 54
124 77
134
138 112
170 141 104 87 50
Five and 10 IL thick sections were incubated for 30 and 40 min.,
1,2, and 3 hours; (a) by increasing the contents of formic acid to 5,10 and 20 % and without formic acid in the developer; (b) by
toning with gold chloride or not. 5 specimens, 2 SDAT and 3 ACA were immunolabeled with antibodies against beta protein and tau protein (kindly provided by Dr. Ogomori, Munchen). The methods, especially the formic acid pretreatment are described elsewhere l ? By help of an ocular grid 10 cortical areas were compared with respect to silverstaining and immunoreaction, counting SP and
Fig. 2. Hippocampus with SP ad NFT in corresponding CA 2 regions: a) silver 10 IL, 1 h toned with gold chloride: b) anti-tau protein; pale stained intact neurons - arrowhead, NFT - arrow; x 400.
Staining of Cerebral Plaques and Tangles· 1047
Fig. 3. Cluster of multipolar NFf-bearing neurons in the prealpha layer of entorhinal allocortex: a) silver, 10 ~, 1 h, no gold chloride; b) anti-tau protein; X 630.
Fig. 4. Angiopathy with perivascular deposits of amyloid, frontal cortex, corresponding areas: a) silver; b) anti· beta protein; x 250.
NFT randomly in corresponding clearly stained cortical fields of 4 x 2 mm and at a magnification of x 100 for SP and x 250 for NFf (see Table 1).
Results
In all 15 cases various numbers of NFT and high amounts of amyloid and neuritic type SP were found; additionally, 6 of these 15 cases (5 ACA and 1 SDAT) showed disseminated deposits of perivascular amyloid (Fig. 4) with patchy staining of the vessels. In 5 cases, apart from SP, various amounts of fibrillar, fluffy amyloid, the diffuse type of Alzheimer plaques (Fig. 5), were seen in the cortex. Comparing the silver stains with the immunohistochemical stains, there was generally a similar topographical distribution pattern of the labeled structures in corresponding sections. By using anti-beta antibodies, which sometimes reacted better with SP, and by using anti-tau antibodies, which gave a distinct reaction with NFT, comparable numbers of SP and NFT were registered in all investigated and counted regions (see Table 1). In particular in the pre-alpha layer of en to rhinal allocortex, clusters of multipolar NFT -bearing neurons were seen,
Fig. 5. Hippocampus with diffuse type of senile plaques: silver 10 ~, 1 h, no gold chloride; x 200.
likewise in silver technique and in anti-tau protein stains (Fig. 3). 5 negative controls did not show SP or NFT in conventional HE and silver stains. In 3 positive cases and 2 negative control cases the one-step silver method was
1048 . E. Reusche compared with von Braunmiihl's method yielding identical positive or negative results. Sections of only 5 ~ thickness as well as incubation times of less than 1 hour were associated with only faint staining of SP and NFT. Increasing the concentration of formic acid caused a loss of argyrophilia of diffuse plaques, pale staining of NFT, and enhanced staining of reticulin structures of capillaries and small vessels. Staining without formic acid gave no reaction after 2 hours. In our experience good and reliable results for SP, fibrillary and perivascular amyloid deposits and NFT are accomplished when 10 ~ thick sections are stained for 1 to 2 hours. Without use of gold chloride the staining intensity of SP and also of mild changes of diffuse plaques is enhanced by the golden brownish colour (Fig. 5); toning with gold chloride makes NFT more visible on a greyish background. Discussion The most obvious advantage of the described silver technique is the simple handling in the daily routine on paraffin sections of any size, compared with the troublesome procedures and unreliable results by silver impregnation on small frozen sections4,9 or on paraffin slices5 • It is easier to perform 11 and it stains reliably both amyloid and neuritic type SP as well as NFT, compared with sensitive silver techniques for demonstration of extracellular amyloid 10 or NFP4, 15. On the one hand the very detailed staining of delicate morphological structures points to a high sensitivity for extracellular (SP and ACA) and intraneuronal (NFT) deposits of A4 protein l8 . On the other hand a high specificity for cerebral amyloid is suggested by the finding that silver staining detects similar numbers of SP and NFT to those detected by immunostaining with anti-beta protein antibodies and with antitau protein antibodies. The existence of clusters of NFT in the allocortex (Fig. 3) has been described by others6 • Even single dark stained NFT can be detected very easily within large groups of normal and intact neurons (Fig. 2). The low concentration of formic acid in the developer is obviously the reason for the applicability of the method in this new field. It allows a careful revealing of argyrophilic structures in SP and NFT. It has been reported that glycolytic pretreatment3 as well as formic acid pretreatment17 enhances immunostaining of cerebral amyloids. However, the latter is said to result in a loss of argyrophilia of diffuse plaques 23 , and this is in agreement with our findings when the concentration of formic acid was increased. The presented silver staining which does distinguish" amyloid" and "neuritic" plaques 7,8 as well as Alzheimer- and non-Alzheimer plaques2,2o,22, may be used as a reliable and inexpensive method for retrospective studies, screening and quantification of cerebral amyloid 13 ,20. Acknowledgements The author wishes to thank Mrs. R. Paustian for expert technical assistence, Prof. Dr. Mehraein and Dr. Ogomori,
Miinchen, for kindly reviewing the manuscript and for providing the antibodies and performing the immunostains, Dr. Diebold for his help in translating the manuscript, and Professor Dr. Lohrs for continuous and encouraging support and critical discussions. All photographs of immunostains were enabled by courtesy of Dr. Ogomori, Miinchen.
References 1 Alzheimer A (1907) Uber eine eigenartige Erkrankung der Hirnrinde. 37. Verslg. Siidwestd. Irrenarzte, Tiibingen, November 1906. Ref Allg Z Psychiatr 64: 146-148 2 Arai H, Lee VM-Y, Otvos L, Greenberg BD, Lowery DE, Sharma SK, Schmidt ML, Trojanowski ]Q (1990) Defined neurofilament, tau, and ~-amyloid precursor protein epitopes distinguish Alzheimer from non-Alzheimer senile plaques. Proc Nat! Acad Sci USA 87: 2249-2253 3 Behrouz N, Defossez A, Delacourte A, Hublau P, Mazzuca M (1989) Methods in laboratory investigation. Alzheimer's disease: glycolytic pretreatment dramatically enhances immunolabeling of senile plaques and cerebrovascular amyloid substance. Lab Invest 61: 576-583 4 Bielschowsky M (1904) Die Silberimpragnation der Neurofibrillen. Einige Bemerkungen zu der von mir angegebenen Methode und den von ihr gelieferten Bildern. ] Psychol Neurol3: 169-189 5 Bodian D (1936) A new method for staining nerve fibers and nerve endings in mounted paraffin sections. Anat Rec 65: 89-97 6 Braak H, Braak E (1985) On areas of transition between entorhinal allocortex and temporal isocortex in the human brain. Normal morphology and lamina-specific pathology in Alzheimer's disease. Acta Neuropathol 68: 325-332 7 Braak H, Braak E, Ohm T, Bohl] (1989) Alzheimer's disease: mismatch between amyloid plaques and neuritic plaques. Neurosci Letters 103: 24-28 8 Braak H, Braak E, Kalus P (1989) Alzheimer's disease: areal and laminar pathology in the occipital isocortex. Acta Neuropathol 77: 494-506 9 von Braunmiihl A (1931) Neue Gesichtspunkte zum Problem der senilen Plaques. Zeitschrift fiir die gesamte Neurologie und Psychiatrie 133: 391-441 10 Campbell SK, Switzer RC, Martin TL (1987) Alzheimer's plaques and tangles: a controlled and enhanced silver staining method. Soc Neurosci (Abstr) 13: 678 11 Cross RB (1982) Demonstration of neurofibrillary tangles in paraffin sections: a quick and simple method using a modification of Palmgren's method. Med Lab Sci 39: 67-69 12 Crocker] (1990) Nucleolar organiser regions. In: Underwood JCE (Ed) Pathology of the Nucleus, Current Topics in Pathology, vol 82, pp 92-149. Springer Verlag, Berlin-Heidelberg-New York 13 Davies L, Wolska B, Hilbich C, Multhaup G, Martins R, Simms G, Beyreuther K, Masters CL (1988) A4 amyloid protein deposition and the diagnosis of Alzheimer's disease: Prevalence in aged brains determined by immunocytochemistry compared with conventional neuropathologic techniques. Neurology 38: 1688-1693 14 Gallyas F (1971) Silver staining of Alzheimer's neurofibrillary changes by means of physical development. Acta Morph Acad Sci Hung 19: 1-8 15 Gallyas F, Wolff JR (1986) Metal-catalyzed oxidation renders silver intensification selective. Application for the histochemistry of diaminobenzidine and neurofibrillary changes. J Histochem Cytochem 34: 1667-1672
Staining of Cerebral Plaques and Tangles . 1049 16 Howell WM, Black DA (1980) Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a I-step method. Experientia 36: 1014-1015 16. Joachim CL, Morris JH, Selkoe DJ (1989) Diffuse senile plaques occur commonly in the cerebellum in Alzheimer's disease. Am J Pathol135: 309-319 17 Kitamoto T, Ogomori K, Tateishi J, Prusiner SB (1987) Methods in laboratory investigation. Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest 57: 230-236 18 Masters CL, Multhaup G, Simms G, Pottgiesser J, Martins RN, Beyreuther K (1985) Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels. Embo Journal 4: 2757-2763 19 Ogomori K, Kitamoto T, Tateishi J, Sato Y, Suetsugu M, Abe M (1989) ~-protein amyloid is widely distributed in the
central nervous system of patients with Alzheimer's disease. Am J Pathol134: 243-251 20 Ogomori K, Mehraein P, Tateishi J, Shin R (1990) Vergleichende Untersuchungen zwischen Morbus Alzheimer und Seniler Demenz vom Alzheimer-Typ unter Anwendung immunhistochemischer Farbungen von beta-Protein und tau-Protein. 35. Jahrestag Dtsch Ges Neuropathol Neuroanat 21 RiischoffJ, Plate K, Bittinger A, Thomas C (1989) Nucleolar organizer regions (NORs). Basic concepts and practical application in tumor pathology. Path Res Pract 185: 878-885 22 Shin R-W, Ogomori K, Kitamoto T, Tateishi J (1989) Increased tau accumulation in senile plaques as a hallmark in Alzheimer's disease. Am J Pathol134: 1365-1371 23 Yamaguchi H, Hirai S, Morimatsu M, Shoji M, Harigaya Y (1988) Diffuse type of senile plaques in the brains of Alzheimertype dementia. Acta Neuropathol 77: 113-119
Received January 21, 1991 . Accepted February 26, 1991
Key words: Silver staining - Senile plaques - Neurofibrillary tangles - Amyloid angiopathy Dr. med Erich Reusche, Institute of Pathology, Medical University Liibeck, Ratzeburger Allee 160, W-2400 Liibeck 1 FRG
Rapid Communication
Silver Staining of Senile Plaques and Neurofibrillary Tangles in Paraffin Sections A Simple and Effective Method E. Reusche Institute of Pathology, Medical University Lubeck, FRG
SUMMARY
A one-step silver staining method using a protective colloidal developer of gelatin and formic acid was originally established for demonstration of argyrophilic nucleolus organizer regions by controlled reduction of silver. We describe here a new application of this silver technique that can easily be performed to demonstrate senile plaques (SP) and neurofibrillary tangles (NFT) on paraffin sections. Preliminary results in ten cases of senile dementia of Alzheimer type and five cases of amyloid congophilic angiopathy showed a reliable demonstration of amyloid and neuritic type SP as well as NFT in all 15 cases. In addition, deposits of perivascular amyloid and areas of fibrillar amyloid, the diffuse type of senile plaques, were revealed. The success seems to depend particularly on the low concentration of formic acid in the developer, which might be the responsible agent for the careful revealing of buried argyrophilic structures in SP and NFT. The staining features were quite similar to those with anti-~ and anti-tau immunostaining. This result suggests a high specificity of this method for extracellular and intraneuronal cerebral amyloid. The detailed staining of delicate morphological structures points to a high sensitivity for cerebral amyloids, senile plaques and neurofibrillary tangles, respectively, by this simple and inexpensive method.
Introduction Since Alzheimer's first description in 19061 of a dementia in a 57-year-old female the characteristic lesions of senile plaques and neurofibrillary tangles have been demonstrated by complicated silver impregnation. The methods of Bielschowsky and von Braunmiihl4 are used on small frozen sections of formalin-fixed material, Bodian's5 and a modified Palmgren's method by Cross ll and are applied on paraffin sections. A one-step silver staining method was originally established for demonstration of argyrophilic nucleolus organizer regions (NOR) on the optical level by the aid of a protective colloidal developer of gelatin and formic acid 16. Recently this sensitive method with a controlled reduction © 1991 by Gustav Fischer Verlag, Stuttgart
of silver has come into use for counting in tumour pathology NOR's in size and number as a possible marker for proliferation or malignancy12,22. While investigating brain tumours by this silver technique, several indistinct, faintly stained senile plaques were found coincidentally in a small tumorous cortical area of a 65-year-old male prompting a careful evaluation of this method.
Material and Methods Between 1985 and 1990 we selected 10 patients with the clinical and/or morphological diagnosis of senile dementia of Alzheimer type (SDAT), 67-88 years of age and brain weights of 0344-0338/9110187 -1 045 $3.50/0
1046 . E. Reusche 950-1120 g; 5 patients with an amyloid congophilic angiopathy (ACA), age 65-87 years; 4 young patients and a 72-year-old
female served as a negative control group; in 5 patients we compared our silver technique with v. Braunmiihl's silver impregnation. Three IL thick sections from different regions (frontal cortex, hippocampus, amygdala and occipital cortex) were stained with Nissl, HE, PAS and congo red stains. The silver staining procedure was performed with slight modifications according to the method of Howell and Black l6 : 1) The colloidal developer was prepared by mixing 1 volume, 2 % gelatin, which had been dissolved in 1 % formic acid (stable for maximum 2 weeks), with 2 volumes of 50 % silver nitrate solution (maturing for several weeks). 2) Deparaffinized sections, 10 IL thick, were incubated with the developer-silver solution in a moist dark chamber, in horizontal position and at room temperature for 60-120 minutes. During the incubation they were covered with a coverslip. Repeated rinsing with distilled water followed. 3) To reduce the brown colour of the background, the slides were subsequently given into 0,1 % gold chloride for 2 minutes, again followed by repeated rinsing. 4) After 2 minutes of incubation in a 3 % sodium-thiosulfate solution the slides were rinsed for 30 minutes in tap water. 5) Finally the sections were dehydrated, cleared and mounted with a synthetic resin (Eukitt, Kindler).
Fig. 1. Low power micrographs of corresponding temporal cortical areas with numerous SP: a) silver (inset: "classical" plaque, x 400); b) anti-beta protein.
Table 1. Counts of senile plaques and neurofibrillary tangles: comparison of silver staining and immuno-reaction in corresponding cortical fields of 8 mm 2 each in five topographically different specimens Senile plaques ~-protein silver
Neurofibrillary tangles silver tau-protein
218 174
202 161
160
52
70 56
80 54
124 77
134
138 112
170 141 104 87 50
Five and 10 IL thick sections were incubated for 30 and 40 min.,
1,2, and 3 hours; (a) by increasing the contents of formic acid to 5,10 and 20 % and without formic acid in the developer; (b) by
toning with gold chloride or not. 5 specimens, 2 SDAT and 3 ACA were immunolabeled with antibodies against beta protein and tau protein (kindly provided by Dr. Ogomori, Munchen). The methods, especially the formic acid pretreatment are described elsewhere l ? By help of an ocular grid 10 cortical areas were compared with respect to silverstaining and immunoreaction, counting SP and
Fig. 2. Hippocampus with SP ad NFT in corresponding CA 2 regions: a) silver 10 IL, 1 h toned with gold chloride: b) anti-tau protein; pale stained intact neurons - arrowhead, NFT - arrow; x 400.
Staining of Cerebral Plaques and Tangles· 1047
Fig. 3. Cluster of multipolar NFf-bearing neurons in the prealpha layer of entorhinal allocortex: a) silver, 10 ~, 1 h, no gold chloride; b) anti-tau protein; X 630.
Fig. 4. Angiopathy with perivascular deposits of amyloid, frontal cortex, corresponding areas: a) silver; b) anti· beta protein; x 250.
NFT randomly in corresponding clearly stained cortical fields of 4 x 2 mm and at a magnification of x 100 for SP and x 250 for NFf (see Table 1).
Results
In all 15 cases various numbers of NFT and high amounts of amyloid and neuritic type SP were found; additionally, 6 of these 15 cases (5 ACA and 1 SDAT) showed disseminated deposits of perivascular amyloid (Fig. 4) with patchy staining of the vessels. In 5 cases, apart from SP, various amounts of fibrillar, fluffy amyloid, the diffuse type of Alzheimer plaques (Fig. 5), were seen in the cortex. Comparing the silver stains with the immunohistochemical stains, there was generally a similar topographical distribution pattern of the labeled structures in corresponding sections. By using anti-beta antibodies, which sometimes reacted better with SP, and by using anti-tau antibodies, which gave a distinct reaction with NFT, comparable numbers of SP and NFT were registered in all investigated and counted regions (see Table 1). In particular in the pre-alpha layer of en to rhinal allocortex, clusters of multipolar NFT -bearing neurons were seen,
Fig. 5. Hippocampus with diffuse type of senile plaques: silver 10 ~, 1 h, no gold chloride; x 200.
likewise in silver technique and in anti-tau protein stains (Fig. 3). 5 negative controls did not show SP or NFT in conventional HE and silver stains. In 3 positive cases and 2 negative control cases the one-step silver method was
1048 . E. Reusche compared with von Braunmiihl's method yielding identical positive or negative results. Sections of only 5 ~ thickness as well as incubation times of less than 1 hour were associated with only faint staining of SP and NFT. Increasing the concentration of formic acid caused a loss of argyrophilia of diffuse plaques, pale staining of NFT, and enhanced staining of reticulin structures of capillaries and small vessels. Staining without formic acid gave no reaction after 2 hours. In our experience good and reliable results for SP, fibrillary and perivascular amyloid deposits and NFT are accomplished when 10 ~ thick sections are stained for 1 to 2 hours. Without use of gold chloride the staining intensity of SP and also of mild changes of diffuse plaques is enhanced by the golden brownish colour (Fig. 5); toning with gold chloride makes NFT more visible on a greyish background. Discussion The most obvious advantage of the described silver technique is the simple handling in the daily routine on paraffin sections of any size, compared with the troublesome procedures and unreliable results by silver impregnation on small frozen sections4,9 or on paraffin slices5 • It is easier to perform 11 and it stains reliably both amyloid and neuritic type SP as well as NFT, compared with sensitive silver techniques for demonstration of extracellular amyloid 10 or NFP4, 15. On the one hand the very detailed staining of delicate morphological structures points to a high sensitivity for extracellular (SP and ACA) and intraneuronal (NFT) deposits of A4 protein l8 . On the other hand a high specificity for cerebral amyloid is suggested by the finding that silver staining detects similar numbers of SP and NFT to those detected by immunostaining with anti-beta protein antibodies and with antitau protein antibodies. The existence of clusters of NFT in the allocortex (Fig. 3) has been described by others6 • Even single dark stained NFT can be detected very easily within large groups of normal and intact neurons (Fig. 2). The low concentration of formic acid in the developer is obviously the reason for the applicability of the method in this new field. It allows a careful revealing of argyrophilic structures in SP and NFT. It has been reported that glycolytic pretreatment3 as well as formic acid pretreatment17 enhances immunostaining of cerebral amyloids. However, the latter is said to result in a loss of argyrophilia of diffuse plaques 23 , and this is in agreement with our findings when the concentration of formic acid was increased. The presented silver staining which does distinguish" amyloid" and "neuritic" plaques 7,8 as well as Alzheimer- and non-Alzheimer plaques2,2o,22, may be used as a reliable and inexpensive method for retrospective studies, screening and quantification of cerebral amyloid 13 ,20. Acknowledgements The author wishes to thank Mrs. R. Paustian for expert technical assistence, Prof. Dr. Mehraein and Dr. Ogomori,
Miinchen, for kindly reviewing the manuscript and for providing the antibodies and performing the immunostains, Dr. Diebold for his help in translating the manuscript, and Professor Dr. Lohrs for continuous and encouraging support and critical discussions. All photographs of immunostains were enabled by courtesy of Dr. Ogomori, Miinchen.
References 1 Alzheimer A (1907) Uber eine eigenartige Erkrankung der Hirnrinde. 37. Verslg. Siidwestd. Irrenarzte, Tiibingen, November 1906. Ref Allg Z Psychiatr 64: 146-148 2 Arai H, Lee VM-Y, Otvos L, Greenberg BD, Lowery DE, Sharma SK, Schmidt ML, Trojanowski ]Q (1990) Defined neurofilament, tau, and ~-amyloid precursor protein epitopes distinguish Alzheimer from non-Alzheimer senile plaques. Proc Nat! Acad Sci USA 87: 2249-2253 3 Behrouz N, Defossez A, Delacourte A, Hublau P, Mazzuca M (1989) Methods in laboratory investigation. Alzheimer's disease: glycolytic pretreatment dramatically enhances immunolabeling of senile plaques and cerebrovascular amyloid substance. Lab Invest 61: 576-583 4 Bielschowsky M (1904) Die Silberimpragnation der Neurofibrillen. Einige Bemerkungen zu der von mir angegebenen Methode und den von ihr gelieferten Bildern. ] Psychol Neurol3: 169-189 5 Bodian D (1936) A new method for staining nerve fibers and nerve endings in mounted paraffin sections. Anat Rec 65: 89-97 6 Braak H, Braak E (1985) On areas of transition between entorhinal allocortex and temporal isocortex in the human brain. Normal morphology and lamina-specific pathology in Alzheimer's disease. Acta Neuropathol 68: 325-332 7 Braak H, Braak E, Ohm T, Bohl] (1989) Alzheimer's disease: mismatch between amyloid plaques and neuritic plaques. Neurosci Letters 103: 24-28 8 Braak H, Braak E, Kalus P (1989) Alzheimer's disease: areal and laminar pathology in the occipital isocortex. Acta Neuropathol 77: 494-506 9 von Braunmiihl A (1931) Neue Gesichtspunkte zum Problem der senilen Plaques. Zeitschrift fiir die gesamte Neurologie und Psychiatrie 133: 391-441 10 Campbell SK, Switzer RC, Martin TL (1987) Alzheimer's plaques and tangles: a controlled and enhanced silver staining method. Soc Neurosci (Abstr) 13: 678 11 Cross RB (1982) Demonstration of neurofibrillary tangles in paraffin sections: a quick and simple method using a modification of Palmgren's method. Med Lab Sci 39: 67-69 12 Crocker] (1990) Nucleolar organiser regions. In: Underwood JCE (Ed) Pathology of the Nucleus, Current Topics in Pathology, vol 82, pp 92-149. Springer Verlag, Berlin-Heidelberg-New York 13 Davies L, Wolska B, Hilbich C, Multhaup G, Martins R, Simms G, Beyreuther K, Masters CL (1988) A4 amyloid protein deposition and the diagnosis of Alzheimer's disease: Prevalence in aged brains determined by immunocytochemistry compared with conventional neuropathologic techniques. Neurology 38: 1688-1693 14 Gallyas F (1971) Silver staining of Alzheimer's neurofibrillary changes by means of physical development. Acta Morph Acad Sci Hung 19: 1-8 15 Gallyas F, Wolff JR (1986) Metal-catalyzed oxidation renders silver intensification selective. Application for the histochemistry of diaminobenzidine and neurofibrillary changes. J Histochem Cytochem 34: 1667-1672
Staining of Cerebral Plaques and Tangles . 1049 16 Howell WM, Black DA (1980) Controlled silver-staining of nucleolus organizer regions with a protective colloidal developer: a I-step method. Experientia 36: 1014-1015 16. Joachim CL, Morris JH, Selkoe DJ (1989) Diffuse senile plaques occur commonly in the cerebellum in Alzheimer's disease. Am J Pathol135: 309-319 17 Kitamoto T, Ogomori K, Tateishi J, Prusiner SB (1987) Methods in laboratory investigation. Formic acid pretreatment enhances immunostaining of cerebral and systemic amyloids. Lab Invest 57: 230-236 18 Masters CL, Multhaup G, Simms G, Pottgiesser J, Martins RN, Beyreuther K (1985) Neuronal origin of a cerebral amyloid: neurofibrillary tangles of Alzheimer's disease contain the same protein as the amyloid of plaque cores and blood vessels. Embo Journal 4: 2757-2763 19 Ogomori K, Kitamoto T, Tateishi J, Sato Y, Suetsugu M, Abe M (1989) ~-protein amyloid is widely distributed in the
central nervous system of patients with Alzheimer's disease. Am J Pathol134: 243-251 20 Ogomori K, Mehraein P, Tateishi J, Shin R (1990) Vergleichende Untersuchungen zwischen Morbus Alzheimer und Seniler Demenz vom Alzheimer-Typ unter Anwendung immunhistochemischer Farbungen von beta-Protein und tau-Protein. 35. Jahrestag Dtsch Ges Neuropathol Neuroanat 21 RiischoffJ, Plate K, Bittinger A, Thomas C (1989) Nucleolar organizer regions (NORs). Basic concepts and practical application in tumor pathology. Path Res Pract 185: 878-885 22 Shin R-W, Ogomori K, Kitamoto T, Tateishi J (1989) Increased tau accumulation in senile plaques as a hallmark in Alzheimer's disease. Am J Pathol134: 1365-1371 23 Yamaguchi H, Hirai S, Morimatsu M, Shoji M, Harigaya Y (1988) Diffuse type of senile plaques in the brains of Alzheimertype dementia. Acta Neuropathol 77: 113-119
Received January 21, 1991 . Accepted February 26, 1991
Key words: Silver staining - Senile plaques - Neurofibrillary tangles - Amyloid angiopathy Dr. med Erich Reusche, Institute of Pathology, Medical University Liibeck, Ratzeburger Allee 160, W-2400 Liibeck 1 FRG