Cf633

Fluorescent antibodies were used for microscopy-based detection of aggregates. The mouse monoclonal anti-α-synuclein Syn211 antibody (Santa Cruz Biotechnology, Inc., Dallas, USA) was labeled with CF633 and CF488A (Biotium, Freemont, USA) and the anti-Aβ IC16 (Heinrich-Heine University, Düsseldorf, Germany) antibody was labeled with CF633 (Biotium, Freemont, USA)^{40 (link),44 (link)}. The labeling process was performed according to the manufacturer’s protocol. The dyes with activated succinimidyl esters were mixed with the antibody in carbonate buffer to react covalently with the amines of the antibody. The labeled antibody was purified using a polyacrylamide bead suspension (Bio-Gel P 30 Gel, Bio-Rad Laboratories, Inc., Hercules, USA). The concentration and degree of labeling were determined according to the manufacturer’s protocol.

For western blots, a fluorescent anti-tau antibody was used. Therefore tau13 (Biolegend, San Diego, USA) was labelled with CF633 (Biotium, Freemont, USA), and the labelling process was performed as described previously [37 (link)]. 2N4R tau recombinant protein samples were prepared in Laemmli buffer (final 1× composition: 20 mM Tris, pH 6.8, 2% SDS, 6% glycerol, 1% β-ME, 0.002% Bromophenol Blue). All samples were heated at 95°C for 5 min and separated using SDS PAGE (15%). Proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane (Thermo Fisher Scientific, Waltham, USA) at 500 mA for 40 min. After a washing step for 15 min in Tris-buffered saline tween buffer (TBS-T) (20 mM Tris, 150 mM NaCl, 0.1% Tween 20), the membrane was blocked for 1 h with 2.5% milk powder/TBS-T. Next, the membrane was washed with TBS-T, 2 × 5 min and in the last step for 15 min. tau13 stocks were 1 mg/ml and were diluted in TBS-T (1:5000). The membrane was incubated with the antibody for 1.5 h. at RT. After a final wash step (2 × 5 min and 1 × 10 min), TBS-T was performed. Detection based on the CF633 fluorescence of the labelled tau13 antibody. Bio-Rad universal hood II and Chemidoc XRS camera and Quantity One 4.6.5 software enabled the visualisation and quantification of the protein bands.