Adaptation of a silver impregnation method for electron microscopic visualization of alzheimer paired helical filaments
✍ Scribed by Clements, J. ;Weiner, L. ;Beitz, A. ;Emory, C. ;Frey, W.
- Publisher
- Wiley (John Wiley & Sons)
- Year
- 1991
- Tongue
- English
- Weight
- 214 KB
- Volume
- 17
- Category
- Article
- ISSN
- 0741-0581
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✦ Synopsis
Neurofibrillar silver-stains were initially developed to delineate normal neuronal architecture (Bielschowsky, 1904) but were soon used for diagnosing the abnormal tangles and plaques in brain tissue from patients with dementia (Alzheimer, 1907). Recently, Campbell and co-workers (1987) have described a silverstaining method for neurofibrillary tangle-containing neurons that is reliable and easy to perform on paraffin or frozen sections. We have adapted this method for use on free-floating vibratome sections and have used it to identify areas of interest to sample at the electron microscopic level. This method can be used in conjunction with routine EM stains to visualize the fine structure of paired helical filaments present in Alzheimer's brain tissue.
Tissue blocks from 3 postmortem Alzheimer's brains were fixed in 3% formalin in 0.1 M phosphate buffered saline containing 2% bovine serum albumin (PBSA) pH 7.4. Fifty-micron vibratome sections from each block were rinsed in 0.1M PBS (pH 7.4) and transferred to water. From water, tissue sections were transferred through the following solutions: 1) 0.6% NH40H (5 1/2l); 2) 2 changes of distilled water (1' each); 3) silver pyridine carbonate (40'); 4) 1% citric acid ( 3 ' ) ; 5) 0.5% acetic acid (3'); 6) physical developer (4 1/2'); 7) 0.5% acetic acid (3'); and 8) water (3'). The silver pyridine carbonate solution was made by mixing 60 ml of 1% AgN03 with 5.4 ml of pyridine, and then adding 45 ml of 1% K2CO3. The physical developer consisted of 5% NazC03 (60 ml) mixed with solution A (45 ml), and solution B (5ml) respectively. Solution A was 0.2% NH4N03 and 1.0% tungstosilicic acid (J.T. Baker Inc.) in water. Solution B consisted of 0.7 ml 37% formaldehyde plus 100 ml of solution A . Solution concentrations and development time were all systematically varied to determine the optimum experimental conditions for EM visualization of silver-stained PHF.
Tissue sections were postfixed in 1% OsO4 for 10 min., embedded in Quetol 651 resin between slides, and examined with a light microscope. Areas containing silver-stained NFT were scribed, thin sectioned and stained with uranyl acetate and lead citrate.
Although PHF were identifiable at the ultrastructural level in osmicated tissue that had not been silver-enhanced (Fig. ) , NFT profiles could not be easily identified in the embedded tissue at the light microscopic level for subsequent thin sectioning. In material enhanced with silver, deposition of silver was limited primarily to the dimeric PHF profiles and was uniform along the length of the profile (Fig. ). The ultrastructural detail seen in silver-enhanced tissue was similar to that seen in silver-01991 WILEY-LISS, INC.