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. 2008 Sep;116(3):277-88.
doi: 10.1007/s00401-008-0409-8. Epub 2008 Jul 15.

Evaluation of alpha-synuclein immunohistochemical methods used by invited experts

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Evaluation of alpha-synuclein immunohistochemical methods used by invited experts

Thomas G Beach et al. Acta Neuropathol. 2008 Sep.

Abstract

The use of alpha-synuclein immunohistochemistry has altered our concepts of the cellular pathology, anatomical distribution and prevalence of Lewy body disorders. However, the diversity of methodology between laboratories has led to some inconsistencies in the literature. Adoption of uniformly sensitive methods may resolve some of these differences. Eight different immunohistochemical methods for demonstrating alpha-synuclein pathology, developed in eight separate expert laboratories, were evaluated for their sensitivity for neuronal elements affected by human Lewy body disorders. Identical test sets of formalin-fixed, paraffin-embedded sections from subjects diagnosed neuropathologically with or without Lewy body disorders were stained with the eight methods and graded by three observers for specific and nonspecific staining. The methods did not differ significantly in terms of Lewy body counts, but varied considerably in their ability to reveal neuropil elements such as fibers and dots. One method was clearly superior for revealing these neuropil elements and the critical factor contributing to its high sensitivity was considered to be its use of proteinase K as an epitope retrieval method. Some methods, however, achieved relatively high sensitivities with optimized formic acid protocols combined with a hydrolytic step. One method was developed that allows high sensitivity with commercially available reagents.

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Figures

Fig. 1
Fig. 1
Flow chart depicting study design
Fig. 2
Fig. 2
Photomicrographs depicting some types of nonspecific staining seen with some of the methods. Corpora amylacea were stained in some methods, including Method 5 (a), glial nuclei were seen in some methods, including Method 4 (b), astrocytes in some methods, including Method 3 (c) and lipofuscin in several methods, including Method 3 (d)
Fig. 3
Fig. 3
Photomicrographs from cingulate gyrus of the same case, showing the range of variation in positive or specific staining obtained by different methods. The four methods depicted represent a range of staining densities, in terms of the total α-synuclein pathology burden. Method 2 (a) was graded at 2.3, Method 3 (b) received a grade of 2.2, Method 4 (c) was graded 1.8 and Method 5 was graded 0.8, on a scale of 0–3 (none, mild, moderate, severe; see “Materials and methods” for details)
Fig. 4
Fig. 4
Graphs comparing nonspecific and specific staining scores obtained by the eight methods and three observers
Fig. 5
Fig. 5
Flow chart for method 10, showing high sensitivity, and performed with all commercial reagents
Fig. 6
Fig. 6
Photomicrographs comparing immunohistochemical staining for α-synuclein in amygdala from the same case, from the two highest scoring methods, Method 2 (a) and Method 6 (b), from the original host lab method (c) and the host lab method adapted by employing proteinase K pretreatment and nickel ammonium sulfate DAB enhancement (d). The total synuclein pathology scores for a–d were, respectively, 2.7, 2.5, 2.1 and 2.8, on a scale of 0–3 (none, mild, moderate, severe; see “Materials and methods” for details)

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