A Red-emitting Fluorescent Probe for Rapid Detection of Protein and Peptide Aggregates in Post-mortem Human Brain Tissue Sections from Patients Diagnosed with Alzheimer’s and Parkinson’s Disease
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Wayne F. Patton1, Dee Shen1 and Kui Tian1
1Enzo Life Sciences, Farmingdale, NY, USA
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INTRODUCTION
Our overall objective was to develop an improved fluorescent probe for detecting aggregated proteins and peptides within cells and tissues. Thioflavin T (ThT) has routinely been employed for determining aggregated protein deposits, both ex vivo and in vitro. However, intrinsic tissue fluorescence of various cellular constituents, such as flavins, reduced NAD(P)H, collagen and elastin substantially overlap with the absorption and fluorescence emission wavelengths of ThT. Additionally, aldehyde fixatives form covalent bonds between adjacent amine-containing groups through Schiff acid–base reactions to generate autofluorescent background with similar spectral properties as ThT. We found that ThT generates fairly high background and weak fluorescent signal in brain tissue sections.
Thus, optimized protocols for the detection of amyloid in tissue sections were developed using a novel red-emitting molecular rotor dye. Frozen and formalin-fixed, paraffin-embed post-mortem human brain tissue (cerebellum) of 5–10 μm in thickness, from patients with Alzheimer’s disease or Parkinson’s disease, as well as adult normal brain tissue were received from a certified tissue vendor who guaranteed that they were collected with informed consent from the donors and their relatives. The fluorogenic red-emitting small molecule probe was found to be suitable for highlighting amyloid-like lesions in neurodegenerative diseases formed by very different constituent peptides and polypeptides, that is β-amyloid peptides and tau proteins associated with Alzheimer’s disease, as well as α-synuclein associated with Parkinson’s disease. Relative to ThT, this novel probe demonstrates significantly higher fluorescence emission intensity enhancement in the presence of amyloid fibrils and lower non-specific background.
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