2.3. Thioflavin T Staining

The ability of both linear and branched KLVFF to prevent Aβ_1-42 fibril self-assembly were initially assessed with thioflavin T (ThT). Briefly, 10 µM Aβ fibrils were mixed with and without KLVFF peptides and added into a glass beaker filled up with silicon oil that it is known to induce amyloid aggregation and therefore able to demonstrate any aggregation inhibitory effect caused by the presence of the blockers. The inhibitor concentrations were set at the same molar concentration (50 µM) of the active principle (KLVFF) rather than of the whole molecules (linear vs. branched KLVFF). The solutions were then left in incubation at 50 °C under continuous agitation (70 rpm) to maximise aggregation conditions and upon a period of 7 days to monitor the effect of the linear and branched blockers upon time. Afterwards, individual samples were mixed with 10 µL of a ThT solution that was previously prepared in deionized water (5 µM) and filtered through a syringe filter with a pore diameter of 0.25 µm (GE Healthcare, Amersham, UK). After 30 min incubation at 37 °C, 150 µL, each solution was added onto tissue culture plate well (Nunc, Rochester, NY, USA) for 10 min at room temperature before being examined by a fluorescence microscopy (Nikon Elipse TE2000U, UK) using a fluorescent dye at the excitation wavelength of 350 nm and an emission wavelength of 438 nm.