Anti tuj1

For immunofluorescence analysis, NPCs were plated on chamber slides (Lab-Tek) or differentiated into neurons on poly-L-ornithine/laminin-coated glass-bottom culture dishes (MatTek). Cells were fixed in 4% (vol/vol) paraformaldehyde for 15 min followed by blocking in phosphate-buffered saline (PBS) containing 8% fetal bovine serum (vol/vol) for 1 h. The primary antibodies were prepared in PBS solution with 2 mg/ml saponin and incubated for 2 h at room temperature or overnight at 4°C. The cells were then washed in PBS and incubated with the corresponding fluorochrome-conjugated secondary antibodies for 1 h. Vectashield mounting medium plus DAPI (Vector Laboratories) was applied to label the nuclei. The following primary antibodies were used: anti-mTOR (Cell Signaling Technology, 2972) 1:100; anti-phospho-mTOR-Ser2448 (Cell Signaling Technology, 5536) 1:100; anti-S6 Ribosomal Protein (Cell Signaling Technology, 2217) 1: 200; anti-phosphoS6-Ser235/236 (Cell Signaling Technology, 2211) 1:100; anti-phospho-4EBP1-Thr37/46 (Cell Signaling Technology, 2855) 1:100; anti-LAMP1 (Developmental Studies Hybridoma Bank, H4A3) 1:200; anti-p62 (BD Biosciences, 610832) 1:100; anti-Tuj1 (Neuromics, MO15013) 1:200; anti-TFEB (MyBioSource, MBS855552) 1:50. The secondary antibodies used were Alexa fluor 488- or 594-conjugated mouse or rabbit (Life Technologies), both at a 1:200 or 1:400 dilution.

For immunofluorescence analysis, cells were fixed in 4% (Vol/Vol) paraformaldehyde for 15 min then blocked in 8% (Vol/Vol) HyClone Fetal Bovine Serum (Thermo Scientific) for 1 h at room temperature. This was followed by incubation with primary antibodies in blocking buffer containing 0.2% Saponin (Sigma-Aldrich) over night at 4°C. Cells were then washed with PBS and incubated with the corresponding fluorochrome-conjugated secondary antibodies (Alexa Fluor 488 or Alexa Fluor 594, Invitrogen) at 1:500 for 1 h at room temperature. Cells were mounted in Vectashield mounting medium containing DAPI (VectorLabs) to label the nuclei. The following primary antibodies were used (all at 1:200 dilution unless otherwise stated): anti-LMX1 (Millipore), anti-Nurr1 (R&D Systems), anti-VMAT2 (Proteintech, 20873-1-AP), anti-GIRK2 (Proteintech, 21647-1-AP and Millipore, AB5200), anti-MAP2 (Millipore, MAB3418), anti-FoxA2 (Millipore, AB4125), anti-mTOR (Cell Signaling, 2972) 1:100, anti-phospho-mTOR-Ser2448 (Cell Signaling, 5536) 1:100, anti-S6 Ribosomal Protein (Cell Signaling, 2217) 1: 200, anti-phosphoS6-Ser235/236 (Cell Signaling, 2211 and 4856) anti-LAMP1 (U. Iowa Developmental Hybridoma Bank, H4A3) 1:100, anti-Tuj1 (Neuromics, MO15013 and BioLegend, 801207), anti-TFEB (Bethyl Laboratories, A303-673A), anti-Tyrosine Hydroxylase (Sigma-Aldirch, T1299), and anti-Bip/Grp78 (abcam, ab21685).