Virchows Archiv A Pathol Anat (1992) 420:519-525 VirchowsArchiv A PathologicalAnatomy and Histopathoiogy 9 Springer-Verlag 1992 Electron microscopic study of paired helical filaments and cerebral amyloid using a novel en bloc silver staining method* E. Reusche 1, K. Ogomori 2, J. Diebold 1, and R. Johannisson 1 1 Institute of Pathology, Medical University, Lfibeck, Federal Republic of Germany 2 Institute of Neuropathology, Ludwig-Maximilian-University, Munich, Federal Republic of Germany Received December 3, 1991 / Received after revision January 28, 1992 / Accepted January 29, 1992 Summary. A one step en bloc silver staining method which was originally established to study nucleolar orga- nizer regions has been applied for the demonstration of both paired helical filaments (PHF) and extracellular cerebral amyloids in semi-thin sections and at the elec- tron microscopic level. The three forms of PHF can be visualized: (1) neurofibrillary tangles are shown in all stages from first appearance in form of intracellular patches of PHF to severely degenerated shadow-like "ghost" tangles; (2) neuropil threads are distinctly stained in great numbers; and (3) PHF are easily de- tected as neuritic components in amyloid plaques. All forms of fibriUar extracellular amyloid structures, i.e. "diffuse", "classical" and" burnt out" plaques, are well demonstrated; congophilic angiopathy reveals amyloid preferentially in arteries and arterioles of the leptomen- inges and cortex ranging from small circumscribed patches to large circumferential amounts with occasional plaque-like condensations or broad loose accumulations of amyloid; perivascular cuffs and laminar subpial de- posits of amyloid are stained as well. At the electron microscopic level all lesions are clearly visible in non uranyl/lead-stained specimens, characterized by varying numbers of silver grains on a pale background. The de- tailed demonstration of structures in archival material, which had been stored in paraffin and re-embedded for electron microscopy, is due to the demonstration of ar- gyrophilic structures by the protective colloidal deve- loper of gelatin and formic acid and to the proteolytic resistance of insoluble PHF and extracellular amyloids in plaques and congophilic angiopathy. Key words: Silver staining Paired helical filaments - Cerebral amyloid - Electron microscopy * Parts of this paper were presented at the Annual Meeting of the German Society for Neuropathology and Neuroanatomy, Dfisseldorf, FRG, 1991 Offprint requests to: E. Reusche, Institute of Pathology, Medical University Lfibeck, Ratzeburger Allee 160, W-2400 Lfibeck 1, Fed- eral Republic of Germany Introduction Novel silver staining methods are used at the light micro- scopical level for a variably selective demonstration of the characteristic lesions in senile dementia of Alzheimer type (SDAT), i.e. neurofibrillary tangles (NFT), neuropil threads (NT) and neuritic plaques (Gallyas 1971; Cross 1982; Gallyas and Wolff 1986), and for extracellular amyloid such as diffuse (Yamaguchi et al. 1990b) and other forms of amyloid plaques (Campbell et al. 1987; Probst et al. 1991). Electron microscopy has revealed NFT (Kidd 1963, 1964; Wisniewski et al. 1976; Miyak- awa et al. 1989) and NT as paired helical filaments (PHF) (Braak et al. 1986; Yamaguchi et al. 1990a) and the fibrillar structure of amyloid has long been identified (Terry et al. 1964; Schlote 1965; Miyakawa et al. 1986). The molecular basis for amyloid has been shown to be a small protein with a 42,43 amino acid sequence, inde- pendently designated as fl- (pleated sheet) protein in ce- rebral vessels (Glenner and Wong 1984) and as amyloid- A4 (4.2 kDa) protein in plaque cores (Masters et al. 1985). Sensitive immunostaining demonstrated the wide distribution of amyloid-flA4-protein in the brains of pa- tients with Alzheimer's disease (Davies et al. 1988; Ogo- mori et al. 1989). Recently we described a new procedure for a simple and effective demonstration of NFT and cerebral amy- loids in paraffin sections (Reusche 1991). Here we con- firm and extend our results by using this silver staining for the demonstration of both PHF and extracellular cerebral amyloids at the electron microscopic level. Materials and methods We selected a total of eight brains, six with SDAT and two with congophilic angiopathy, four of which came from our recently de- scribed autopsy series (Reusche 1991). Small pieces of paraffin- embedded tissue were taken from regions where light microscopy had revealed severe morphological changes. The samples were de- paraffinized using xylene and rehydrated in a graded ethanol series. The silver staining procedure was performed according to Plo- ton et al. (1982) with slight modifications: deparaffinized and re-