Alexa dye labeled secondary antibodies

Cells were grown on poly-D-lysine coated 12 mm round coverslips and fixed in 4% formaldehyde solution. Cells were washed 3 × 5 min in PBS/Ca²⁺/Mg²⁺, then blocked in antibody buffer (PBS, 0.5% Triton X-100, 1 mM EDTA, 0.1% BSA, 0.05% NaN₃) with 10% normal goat serum. Primary antibodies were applied and incubated for 1 h at room temperature. Primary antibodies used were: pTau = AT180 (Thermo Fisher), TOE1 = Thermo Fisher (Catalog# PA5-30948), PARN = Protein Tech (Catalog# 13799-1AP), PAN2 = Protein Tech (Catalog# 16427-1AP), CNOT6 = Cell Signal (Catalog# 13415). Cells were washed 3 × 5 min in PBS/Ca²⁺/Mg²⁺, then re-blocked for 10 min. Appropriate Alexa dye-labeled secondary antibodies (Invitrogen) were applied and incubated for 20 min at room temperature. Cells were again washed 3 × 5 min in PBS/Ca²⁺/Mg²⁺, counterstained with 300 nM DAPI and mounted with ProLong Gold antifade (Molecular Probes). Microscopy was performed on a Delta Vision microscope (GE, Inc) using a 100× oil immersion objective, a sCMOS camera, and 2 × 2 binning. Image analysis was performed using softWoRx 6.0 Beta software (GE, Inc).