Grow STC-1 cells on 12 mm glass coverslips treated with poly-D-lysine or Geltrex™.

Fix cells with 4 % paraformaldehyde 0h 15m 0s

Wash cells with PBS 2 x 0h 5m 0s

Aspirate and discard PBS and freeze cells < -20°C

Thaw frozen 24-well plates containing fixed cells grown on glass coverslips

Wash cells with PBS 0h 5m 0s

Quench with 0.3% H₂O₂ 0h 5m 0s

Wash cells with PBS 2 x 0h 5m 0s

Block with 10 % goat serum in PBS containing 0.005 % Triton™ X-100 and 0.05 % thimerosal1h 0m 0s

Incubate with rabbit anti-alpha-synuclein (1:500) (Abcam, ab212184) in 10 % goat serum containing 0.005 % Triton™ X-100 and 0.05 % thimerosal (block buffer) 20h 0m 0s

Wash with PBS 4 x 0h 5m 0s

Incubate with biotinylated goat anti-rabbit IgG (1:500) (Stratech, 111-065-144-JIR) in 2 % goat serum containing 0.005 % Triton™ X-100 and 0.05 % thimerosal (antibody buffer) 2h 0m 0s

Wash with PBS 4 x 0h 5m 0s

Incubate with 1:250 streptavidin-HRP (Roche, 11089153001) for 1 hour in antibody buffer1h 0m 0s

Wash with PBS 6 x 0h 5m 0s

Visualise staining by incubation with PBS containing 0.05 % (w/v) 3,3′-Diaminobenzidine/0.015 % (v/v) H₂O₂/0.05 % (w/v) nickel ammonium sulphate for ≤ 0h 3m 0s

Rinse for > 0h 5m 0s in running tap water

Remove coverslips from wells and mount on microscope slides with Hydromount™ (National Diagnostics)