Immunocytochemistry and ImageJ quantification
Cells were grown on glass coverslips for 24 h prior to indicated treatments and fixation with 4% paraformaldehyde (Sigma-Aldrich P6148). The fixed cells were permeabilized in –20°C methanol and blocked in 5% normal goat serum containing 0.05% sodium azide (Life Technologies 50062Z) in preparation for immunostaining. Mouse anti-FUS (Santa Cruz 373698) and custom-produced rabbit antibodies against phosphorylated FUS (Genscript) antibodies were used as primary antibodies to probe the fixed cells. AF488 and AF586 conjugated secondary antibodies (Southern Biotech 1030-30; Life Technologies A11011) were used to detect the primary antibodies, and DAPI-containing mounting media (Invitrogen P36931) was used to stain the nuclei and mount the coverslips to glass slides. The Zeiss 700 confocal microscope was used to view and image the prepared slides. Biological replicates (n) were produced from separately cultured cells.
Quantification was performed using Fiji (Schindelin et al., 2012). For each immunofluorescence microscopy image shown, between 5 and 10 cells were quantified per biological replicate for statistical analysis. Quantification of subcellular localization was done using the Raw Integrated Density measurement. Nuclear and cytoplasmic areas were mapped under the highest maximum threshold setting and a minimum threshold setting of 10 and 1, respectively. Quantitative measurements were made without applied thresholds. Knockdown experiments were quantified using the mean gray value measurement with the same mapping techniques. All data are displayed as the mean value with error bars representing 95% confidence intervals using GraphPad Prism version 7.00 for Windows (GraphPad Software).