Rabbit anti th sc 14007

iPSC-derived cells were fixed with 4% paraformaldehyde (PFA) in Tris-buffered saline (TBS) buffer for 20 min and blocked in 0.3% Triton X-100 (Sigma-Aldrich, Madrid, Spain) with 3% donkey serum for 2 h. In the case of α-SYN and LC3 staining, Triton X-100 was kept at 0.01% for the blocking and antibody incubation steps. The following primary antibodies were used: mouse anti-α-SYN (610787; 1:500) (BD Biosciences, Madrid, Spain), rabbit anti-TH (sc-14007; 1:500) (Santa Cruz Biotechnology, Madrid, Spain), chicken anti-GFP (1020; 1:250) from Aves Labs (Cosmo Bio, AbBcn S.L., Bellaterra, Spain), rabbit anti-cleaved Caspase3 (9664; 1:400) (Cell Signaling) and LC3B (2775; 1:100) (Cell Signaling). Images were acquired using a Leica SP5 confocal microscope.

For immunofluorescence, cells were fixed for 20 minutes with 4% paraformaldehyde (EM Sciences) in PBS. Permeabilization and blocking were performed in a single step using PBS (Lonza) supplemented with 0.1% Triton X-100 (Sigma), 10% FCS (GE Healthcare) and 1% BSA. Primary antibodies were applied overnight at 4°C in PBS containing 0.1% BSA. Subsequently, cells were incubated with secondary antibodies for 1 h at room temperature and ultimately washed 3x, including a Hoechst (Thermo Fisher Scientific) counterstaining for nuclei in the second washing step. Primary antibodies used in this study included: goat anti-Sox2 sc-17320 1:200 and mouse anti-Oct4 sc-5279 1:200 (both Santa Cruz), mouse anti-SSEA4 MC-813-70 1:200 (DSHB), goat anti-Sox1 AF3369 1:400, mouse anti-Nestin MAB1259 1:500, (both R&D Systems), rabbit anti-TH sc-14007 1:300 (Santa Cruz), mouse anti-Tuj1 MMS-435P 1:1000 (BioLegend). For fluorescence microscopy analysis, secondary antibodies conjugated to Alexa-488, 568 or 647 (1:1000, Thermo Fisher Scientific) were used. Cells were imaged on an inverted Apotome Zeiss Axio/Observer Z1 fluorescence microscope.