Duolink in situ probemaker plus

Cell lines plated on 96‐well plates were fixed in pure ice‐cold methanol for 7 min at −20°C, followed by three cell culture‐grade PBS washes. PLA assay was performed using custom labeled antibodies and the Duolink In Situ Detection Reagents Red (Sigma‐Aldrich, DUO92008) as described by the manufacturer but with slight modifications. In brief, antibodies of choice (anti‐TDP‐43, anti‐GFP, and anti‐HA in Appendix Table S3) in a carrier‐free buffer were labeled with MINUS and PLUS probes using the Duolink In Situ Probemaker MINUS (Sigma‐Aldrich, DUO92010) and Duolink In Situ Probemaker PLUS (Sigma‐Aldrich, DUO92009) according to the manufacturer's instructions. After blocking for at least 15 min with blocking solution at room temperature, probe‐conjugate antibodies were incubated overnight at 4°C. After two washes of 5 min each with buffer A (10 mM Tris, 150 mM NaCl 0.05% Tween 20), ligation reaction was performed as indicated by the manufacturer. After an additional two washes of 5 min each with buffer A, amplification reaction was performed as indicated by the manufacturer. The reaction was quenched with buffer B (100 mM Tris, 100 mM NaCl). If required, secondary antibodies were incubated in cell culture‐grade PBS overnight at 4°C, followed by DAPI counterstaining and imaging in cell culture‐grade PBS.