Adult heads were fixed with 4% paraformaldehyde in phosphate buffered saline (PBS) for 45 min at room temperature (RT). Fly brains were dissected in PBS containing 0.1% Triton X-100 (PT). Brains were blocked in 10% normal goat serum for 1 h in PT and incubated with antibody 6E10 at 4°C overnight. After incubation with Cy3-labeled anti-mouse antibody (Jackson InmunoResearch, West Grove, PA, USA) for 2 h at RT, brain tissue was stained with DAPI, washed with PBS and mounted in PBS containing 80% glycerol. For amyloid fibril staining, brains were incubated in 50% ethanol containing 1% thioflavine S (ThS; Sigma, St.Louis, MO, USA) overnight at 4°C. Samples were washed with PBS containing 50% ethanol and mounted in 80% glycerol. Brain samples from a transgenic mouse carrying the “Swedish” mutation of amyloid precursor protein (Tg2576) were used as positive controls. Images were captured with a Zeiss LSM 510 Meta Confocal microscope.
Thioflavine s ths
Manufactured by Merck Group
Sourced in United States
Thioflavine S (ThS) is a fluorescent dye commonly used in research applications. It functions as a molecular probe, selectively binding to specific structures or compounds, allowing their detection and visualization. The core function of Thioflavine S is to serve as a labeling agent for analytical and imaging purposes.
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2 protocols using thioflavine s ths
1
Immunofluorescent Staining of Amyloid Fibrils in Adult Fly Brains
2
Live-Cell Imaging of Mitochondria and Protein Aggregation
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The live cell staining was performed to visualize the mitochondria and for detection of the protein aggregation process. MitoTracker®RED FM (MTR, Invitrogen, M22425) was used to display the mitochondrial network with low specificity to the mitochondrial transmembrane potential. Alternatively, the tetramethylrhodamine (TMRM, Sigma, T5428) fluorescence probe was used to monitor transmembrane potential of mitochondria. Cells were stained in fresh growth medium containing MTR or TMRM at a final concentration of 100 nM (100 mM stock in DMSO) for 30 min at 37 °C in humidified 5 % CO 2 atmosphere. Subsequently, the cells were washed three times with PBS. For regeneration, the cells were incubated for 30 min in fresh pre-warmed growth medium without dye at 37 °C and immediately analyzed. The same staining conditions were maintained for detection of cytoplasmic inclusions in living cells by Thioflavine S (ThS, Sigma, T1892) with a final concentration of 0.0005 % m/v. Naturally occurring endo-fluorophore NADH has also been used to monitor intracellular changes by confocal microscopy.
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