Labeling Tau Oligomers with Alexa Fluor 488

Prepared recombinant TauO were provided by Dr. Rakez Kayed’s laboratory. They were produced and characterized following established and published protocols [33 , 34 (link)] and labeled (TauO488) as previously described [35 (link)–37 (link)]. Briefly, 1 mg of Alexa Fluor™ 488 NHS Ester Succinimidyl Ester (cat# A20000, Thermo Fisher Scientific) was dissolved in 0.1 M sodium bicarbonate to a final concentration 1 mg/ml, pH 8.3. The dye was then incubated with TauO in a 1:4 (w/w) ratio, rotating overnight at 4 °C on an orbital shaker. The following day, the solution was centrifuged (30 min, 15,000 × g) using 10-kDa Amicon Ultra‐0.5 ml Centrifugal Filter Units (cat# UFC501024; EMD Millipore, Burlington, MA, USA) to remove unbound dye. TauO were then washed with 1 × phosphate-buffered saline (PBS) until the flow-through solution was clear. The filter compartment was then flipped and centrifuged to collect the concentrate (2 min, 1000 × g). Oligomer concentrations were then quantified with the Pierce™ BCA Protein Assay Kit (cat# 23227, Thermo Fisher Scientific) and used for flow cytometry and immunofluorescence analyses.