Ab27766

Undifferentiated SH-SY5Y cells, seeded on coverslips (No. 1.5) were fixed in 4% PFA for 10 min, blocked with 20% SeaBlock containing 0.1% Triton X-100 for 1 h at 21°C, and stained with anti-α-synuclein (ab27766; Abcam) overnight at 4°C. Cortical neurons were treated with α-Syn fibrils or vehicle control for between 72 h and 14 days,^{23 (link)} fixed and stained for anti-PIP5K1γ (gift from Dr. Pietro DiCamilli^{38 (link)}). Cells were incubated for 1 h in Alexa Fluor 647 donkey anti-rabbit (A31573; 1:1,000; Invitrogen) or Alexa Fluor 647 goat anti-mouse (A21236; 1:1000; Invitrogen) secondary antibody in blocking solution. Coverslips were then mounted onto glass depression slides (neoLab, Heidelberg, Germany) with a cysteamine (MEA)-catalase/glucose/glucose oxidase (GLOX) imaging buffer containing TN buffer (50 mM Tris pH 8.0, 10 mM NaCl), a GLOX oxygen scavenging system (0.56 mg mL– 1 glucose oxidase, 34 μg mL–1 catalase, 10% w/v glucose) and 100 mM MEA. Twinsil dental glue (Picodent, Wipperfürth, Germany) and aluminum tape (T205–1.0 - AT205; Thorlabs Inc., Newton, NJ, USA) were used to hold the coverslip in place on the slide and to exclude oxygen. Images were captured using a Leica Infinity TIRF super-resolution microscope equipped with a 163×1.49 NA TIRF oil immersion objective and a Hamamatsu orca flash 4.0 camera. Particle Analysis in ImageJ was conducted using 20 nm pixel size.