Rabbit anti asyn

Twenty-four or forty-eight hours after transfection, cells (HEK or H4) were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 minutes at room temperature (RT), followed by a permeabilization step with 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 20 minutes at RT. After blocking in 1.5% normal goat serum (PAA, Cölbe, Germany)/DPBS for 1 hour, cells were incubated with primary antibody. Primary antibodies used were: mouse anti-ASYN (1∶1000, BD Transduction Laboratory, New Jersey, USA) or rabbit anti-ASYN (1∶1000, Abcam, Boston, USA), rabbit anti-LAMP-1 (1∶1000, Abcam, Boston, USA), anti-Giantin (1∶1000, Abcam, Boston, USA) for 3 hours or overnight and secondary antibody (Alexa Fluor 488 donkey anti-mouse IgG and/or Alexa Fluor 555 goat anti rabbit IgG, (Life Technologies- Invitrogen, Carlsbad, CA, USA) for 2 hours at RT. Finally, cells were stained with Hoechst 33258 (Life Technologies- Invitrogen, Carlsbad, CA, USA) (1∶5000 in DPBS) for 5 minutes, and maintained in PBS for epifluorescence microscopy.

After transfection, cells were fixed with 4% paraformaldehyde at room temperature (RT), followed by a permeabilization with 0.5% Triton X-100 (SigmaAldrich, St. Louis, MO, USA). The cells were blocked in 1.5% normal goat serum (PAA, Cölbe, Germany)/1xPBS (1.37 M NaCl, 27 mM KCl, 101.4 mM Na₂HPO₄7^.H₂O, 16.7 mM KH₂PO₄), and then incubated with primary antibody. Primary antibodies used in this study were: mouse Syn1 (1:1000, BD Transduction Laboratory, New Jersey, USA) or rabbit anti-aSyn (1:1000, Abcam, Boston, USA), anti-Giantin (1:1000, Abcam, Boston, USA), aSyn-S129 1:1000 (Wako Chemicals USA, Inc., Richmond, USA) overnight, and secondary antibody (Alexa Fluor 488 donkey anti-mouse IgG and/or Alexa Fluor 555 goat anti rabbit IgG, (Life Technologies- Invitrogen, Carlsbad, CA, USA)) for 2 h at RT. Cells were finally stained with Hoechst 33258 (Life Technologies- Invitrogen, Carlsbad, CA, USA) (1:5000 in DPBS) for 5 min, and maintained in 1xPBS for imaging.