Mab1637

Cells were fixed for 20 min at room temperature with 4% paraformaldehyde, washed with PBS, and blocked for 45 min at room temperature with PBS containing 10% normal goat serum and 0.03% Triton X-100. Cells were then probed with primary antibodies against Oct4 (Oct4; monoclonal, 1:100, Abcam sc-9081), Nanog (Nanog; monoclonal, 1:200, Abcam ab80892), tubulin beta III (Tuj1; monoclonal, 1:1000, Millipore MAB1637), SMA (SMA; monoclonal, 1:200, Abcam ab7817), and Sox17 (Sox17; polyclonal, 1:200, R&D systems AF1924). Finally, cells were labeled with secondary antibodies conjugated to Alexa Fluor 488 or 568 (Molecular Probes, Eugene, OR, USA), following specifications of the manufacturer.

The NPCs and neurons grown on cover slips were fixed in 4% paraformaldehyde in 1x PBS for 1 hr at 4°C and washed twice with 1x PBS. The fixed cells were incubated in 0.3% Triton X 100 for 30 min to 1 hr, and treated with 50 mM NH₄Cl in 1x PBS for 15 min followed by a 1x PBS wash. The cells were blocked for 1 hr at RT in blocking buffer (5% goat serum and 0.4% Triton X 100 in PBS). Primary antibodies diluted in the Blocking buffer were applied to the cells and incubated overnight at 4°C. After PBS washing (3x 10 min), the secondary antibodies, goat anti-mouse or/and goat anti rabbit conjugated with Alexa Fluor 488 or Alexa Fluor 633 (1: 1500) in blocking buffer were applied and incubated for 1 hr at RT. Following 1x PBS washes (3x 10 min), the cells were mounted with VECTASHIELD mounting medium containing DAPI (Vector H1200). The signals were observed under Zeiss Apotome fluorescence microscope. The antibodies used: rabbit anti-nestin (1:125; Millipore, AB5922), rabbit anti-sox2 (1: 100; Millipore, AB5603), rabbit anti-Map2 (1:200; Millipore, AB5622), mouse anti-α-synuclein (1:10; Invitrogen, 18-0215), mouse anti-synaptophysin (1:100; Millipore, MAB5258), rabbit anti-Tyrosine hydroxylase (1:100; Chemicon, AB152), mouse anti-Tuj1 (1:100; Millipore, MAB1637), mouse anti-Lamp1 conjugated with Cy5 (1:100; Abcam, ab25222), and rabbit anti-human GCase (1:100) [45 (link)].

Cells were washed three times with sterile PBS and then fixed using 4% PFA for 20 min. After washing, cells were blocked 0.3% Triton-X and 1% goat serum in PBS for 1 h. Primary antibodies specific for Synapsin1 (1:500; ab254349; Rabbit; Abcam, Cambridge, MA, USA), NeuN (1:500; ab104225; Rabbit; Abcam, Cambridge, MA, USA), Beta-III Tubulin (1:500; MAB1637, Mouse; Kenilworth, NJ, USA), MAP2 (1:1000; Chicken; ab5392; Abcam, Cambridge, MA, USA), TBR1 (1:200; ab183032; Rabbit; Abcam, Cambridge, MA, USA), GFAP (1:500; ab4674; Chicken; Abcam, Cambridge, MA, USA), and Ki67 (1:500; ab245113; Mouse; Abcam, Cambridge, MA, USA) were incubated overnight. After washing, secondary antibodies (chicken 555, rabbit 488, mouse 647; Abcam, Cambridge, MA, USA) were incubated for 2 h. This was followed by 10 min of DAPI Staining Solution in PBS (1:1000, ab228549, Abcam, Cambridge, MA, USA) after which point slides were cover-slipped with ProLong Gold Antifade Mountant (Thermo Fisher Scientific, Waltham, MA, USA) mounting media and allowed to dry for 48 h.