Mouse anti fus

Proximity ligation assays (PLAs) were performed essentially as the manufacture instructions (Sigma-Aldrich). Briefly, neurons were fixed in 4% paraformaldehyde in PBS and probed with mouse anti-FUS (1:200, Proteintech) and anti-SNPH (1:200, Proteintech), and signals developed using a Duolink In Situ Orange kit (Sigma-Aldrich). Following PLAs, neurons were immunolabeled for chicken anti-MAP2 (1:1000, AbCam). Images were taken at × 60 (oil) on a Nikon Ti-E Two Camera microscope. Images were analysed in ImageJ and positive puncta counted using the cell counting tool. Five somas for each image were analysed for the soma count and 3 different neurites for each of five cells per image were analysed with three biological replicates carried out.

Primary neurons were fixed in 4% paraformaldehyde for 10 min and then ice cold methanol for 10 min before being probed with selected primary antibodies overnight at 4 °C. Antibodies were used as follows rabbit anti-synaptophysin I (1:300, Synaptic Systems), rabbit anti-FUS (1:300, Sigma), mouse anti-FUS (1:200, Proteintech), mouse anti-PSD95 (1:500, AbCam), mouse anti-HA (1:1000, CoVance), chicken anti-MAP2 (1:1000, AbCam), mouse anti-FUS (H6) (1:200, Santa Cruz), rabbit anti-SNPH (1:200, ProteinTech). Goat anti-rabbit, anti-mouse or anti-chicken Alexaflour 488/564/640 secondary antibodies (1:500, Invitrogen) were added the following day for two hours at room temperature in the dark. DAPI (4ʹ,6-diamidino-2-phenylindole (Sigma) counterstaining (1.25 μg/ml) was added as a nuclear stain before cover slips were mounted.