X-34, A Fluorescent Derivative of Congo Red: A Novel Histochemical Stain for Alzheimer's Disease Pathology

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Abstract
X-34, a lipophilic, highly fluorescent derivative of Congo red, was examined as a histochemical stain for pathological changes in Alzheimer's disease (AD). X-34 intensely stained neuritic and diffuse plaques, neurofibrillary tangles (NFTs), neuropil threads, and cerebrovascular amyloid. Comparison to standard methods of demonstrating AD pathology showed that X-34 correlated well with Bielschowsky and thioflavin-S staining. X-34 staining of NFTs correlated closely with anti-TAU antibody staining. A 1:1 correspondence of X-34 and anti-Aβ antibody staining of plaques and cerebrovascular amyloid was observed. Both X-34 and thioflavin-S staining were eliminated by formic acid pretreatment, suggesting that β-sheet secondary protein structure is a necessary determinant of staining. X-34 may be a general amyloid stain, like Congo red, because it also stains systemic amyloid deposits due to λ-light chain monoclonal gammopathy. In conclusion, X-34 is a highly fluorescent marker for β-sheet structures and intensely labels amyloid plaques, NFTs, neuropil threads, and vascular amyloid in AD brains. It can be used with both paraffin-embedded and frozen tissues as well as in combination with immunohistochemistry for double labeling. The intensity of staining and the simplicity and reproducibility of the technique suggest that it may be a useful addition to the standard techniques for evaluation of AD neuropathology.