Chicken anti smi32

For immunofluorescence staining, cells were washed twice with PBS without Ca²⁺/Mg²⁺ (LifeTechnologies) and fixed with 4% PFA in PBS for 10 min at room temperature. PFA was aspirated and cells were washed three times with PBS. Fixed cells were first permeabilized for 10 min in 0.2% Triton X solution and subsequently incubated for 1 h at RT in blocking solution (1% BSA, 5% donkey serum, 0.3 M glycine and 0.02% Triton X in PBS). Following blocking, primary antibodies were diluted in blocking solution and cells were incubated with primary antibody solution overnight at 4 °C except for the γH2A.X antibody which was kept for only 2 h at room temperature on the fixed material. The following primary antibodies were used: chicken anti-SMI32 (1:10,000, Covance), mouse anti-FUS (1:5000, Sigma-Aldrich), rat anti-meFUS (1:100^{5 (link)},), rabbit anti-beta-III-Tubulin (1:3000, Covance), mouse anti-Hb9 (1:100 Development studies Hybridoma Bank), rabbit anti-Islet (1:500, Abcam), mouse anti-yH2A.X (1:500 Millipore), rabbit anti-ChAt (1:500, Chemicon), rabbit anti-53BP1 (1:1000, Novusbio). Nuclei were counter stained using Hoechst (LifeTechnologies).

For immunofluorescence staining, cells were washed twice with phosphate-buffered-saline (PBS) without Ca²⁺/Mg²⁺ (LifeTechnologies, Carlsbad, CA, USA) and fixed with 4% paraformaldehyde in PBS for 10 min at room temperature. Paraformaldehyde was aspirated and cells were washed three times with PBS. Fixed cells were first permeabilized for 10 min in 0.2% Triton X solution and subsequently incubated for 1 h at room temperature in blocking solution (1% bovine serum albumin, 5% donkey serum, 0.3M glycine (Carl Roth GmBH & Co. KG, Germany) and 0.02% Triton X-100 (Thermo Fisher Scientific, Waltham, MA, USA) in PBS). Following blocking, primary antibodies were diluted in blocking solution and cells were incubated with primary antibody solution for 12 h at 4 °C. The following primary antibodies were used: chicken anti-SMI-32 (1:10.000, Covance, Princeton, NJ, USA), mouse anti-FUS (1:5000, Sigma Aldrich, St. Louis, MO, USA), rabbit anti-beta-III-Tubulin (1:3000, Covance, Princeton, NJ, USA), mouse anti-GM-130 (1:200, BD Pharmingen, Franklin Lakes, NJ, USA), mouse anti-SOD1 (1:100, Cell Applications Inc., San Diego, CA, USA), rabbit anti-TDP-43 (1:400, Abcam, Cambridge, UK), rabbit anti-GFAP (1:1000 Chemicon, Temecula, CA, USA), mouse anti-vimentin (1:100, Sigma Aldrich, St. Louis, MO, USA). The nuclei were counterstained using Hoechst 33342 (LifeTechnologies, Carlsbad, CA, USA).