647 conjugated antibody against tau13

Cell immunostaining was performed as previously described with slight modification [25 (link),26 (link)]. Briefly, N2A−Htau cells were plated on Matrigel-coated 35 × 10 mm No 1.5 glass-bottom dishes (Eppendorf) and grown to 60% confluence prior to VPS35 siRNA transfection. After a 72 h incubation, cells were washed with PBS and fixed in 4% paraformaldehyde in PBS for 20 min at RT. After washing several times with PBS and permeabilizing with 0.2% Triton X-100 in PBS, cells were incubated in blocking solution (5% normal donkey serum in PBS) for 1 h at RT followed by an O/N incubation at 4 °C with a combination of primary antibodies prepared in 2% blocking solution in PBS against VPS35 (1:250, Abcam), Tau13 (1:200, Biolegend), PHF13 (1:20, Thermo), AT270 (1:100, Thermo), and MC-1 (1:20, provided by Peter Davies). After several washings with PBS, cells were incubated for 1 h at RT with secondary Alexa Fluor 568-conjugated antibody against VPS35 and 647-conjugated antibody against Tau13, AT270, PHF13 or MC-1 (all at 1:250; Abcam). Cells were then stained with DAPI, washed with PBS and subsequently held at 4 °C in the dark until imaging.