Primary anti sod 1

SOD-1 aggregates was identified by non-denaturing polyacrylamide gel electrophoresis. Proteins (50µg) were separated by non-denaturing electrophoresis. Samples were diluted in a Sample Buffer 5X without SDS, and also the electrophoresis buffer is without SDS, and then blotted onto a nitrocellulose membrane. Membrane was blocked with 3% bovine serum albumin in T-TBS and incubated for 1 h and 30 min at room temperature with primary anti-SOD-1 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) and for 1 h at room temperature with secondary antibody horseradish peroxidase-conjugated anti-mouse IgG (1:5000; Sigma–Aldrich, St Louis, MO, USA). Membrane was developed with the Super Signal West Pico chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA), acquired with Chemi-Doc MP (Bio-Rad) and analyzed using Image Lab software (Bio-Rad).

For Western blot, 40 µg of proteins (CTR young, old and DS with and without AD) were separated by 12% SDS–PAGE and blotted onto a nitrocellulose membrane (Bio-Rad, Hercules, CA, USA). Membrane was blocked with 3% bovine serum albumin in T-TBS and incubated for 1 h and 30 min at room temperature with primary anti-SOD-1 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA) and for 1 h at room temperature with secondary antibody horseradish peroxidase-conjugated anti-mouse IgG (1:5000; Sigma–Aldrich, St Louis, MO, USA). Membrane was developed with the Super Signal West Pico chemiluminescent substrate (Thermo Scientific, Waltham, MA, USA), acquired with Chemi-Doc MP (Bio-Rad) and analyzed using Image Lab software (Bio-Rad).