Characterization of Tau Fibril Binding Probes

Detailed information on the probes and chemical compounds is listed in Suppl. Table 1 [60 (link), 61 (link)]. Recombinant K18 4R tau was expressed and produced by Escherichia coli as described previously [62 , 63 ] (Supplementary material). Details on recombinant K18 tau fibril production and characterization are in SFig. 1 and supplementary methods. The absorbance of the compounds was measured with a spectrofluorometer. Thioflavin T assays against K18 tau fibrils using a fluorometer (Fluoromax 4, Horiba Scientific, Japan) were performed as described previously [62 ], with two independent experiments and three technical replicates. PBB5 (excitation peak 630 nm, concentration 1.6 μM) was dissolved in Milli-Q H₂O or dimethyl sulfoxide and further diluted in 1 × PBS (Gibco). PBB5 was then mixed with 5 μL of tau K18 fibril solution in a 45-µL quartz cuvette (quartz SUPRASIL Ultra Micro Cell, Hellma). The solution was incubated for 1 min at room temperature and resuspended, and fluorescence was measured with a spectrofluorometer using the corresponding excitation wavelength.