M4403

Tissue samples were fixed in 4% buffered formaldehyde, dehydrated, embedded in paraffin, and sectioned at 2 µm. H&E staining was performed according to standard laboratory protocols. Immunohistochemical stainings were performed on a Ventana BenchMark XT system (Roche Diagnostics). The following primary antibodies were used: SALL4 (ab57577, abcam, 1:50), Ki67 (SP6, Cell Marque, 1:750), SOX2 (ab97959, abcam, 1:1000), MAP2C (M4403, Sigma–Aldrich, 1:3000), CD56 (MSK006, Zytomed, 1:2000), Synaptophysin (M7315, Dako, 1:500). As a chromogen, 3,3ʹ-diaminobenzine (DAB) was used.

For immunocytochemistry of NPCs, cells were fixed with 4% paraformaldehyde in PBS for 10 min at room temperature (RT). After blocking nonspecific staining in PBS containing 10% normal goat serum (NGS), 1% bovine serum albumin (BSA) and 0.1% Triton X-100 for 45 min at RT, cells were incubated with the primary antibodies overnight at 4°C. Primary antibodies were diluted in PBS containing 1% NGS, 1% BSA and 0.1% Triton X-100. All primary antibodies used in the study are commercially available and well characterized. We used primary antibodies recognizing SOX2 (1:25, MAB 2018; R&D Systems), Nestin (1:200, SC-20978; Santa Cruz Biotechnologies), DCX (1:500, AB2253; Millipore), and MAP2 (1:2500, M4403; Sigma). Secondary antibodies were applied in PBS containing 1% BSA for 45 min at RT. Secondary antibodies were goat anti-chicken Alexa Fluor 488 (1:500, A-1139, Invitrogen), anti-rabbit Alexa Fluor 546 (1:500, A11003; Invitrogen), and anti-mouse Alexa Fluor 635 (1:500, A-31574; Invitrogen). After final washes, the cell nuclei were counterstained with Vectashield mounting media containing DAPI (Vector Laboratories).

Cells were grown at density of 6 × 10⁴ on glass coverslips 12 mm diameter round. Cells were fixed at different time points after differentiation (3, 7 and 10 days) with 4% formaldehyde solution for 15 min at room temperature. Cells were then washed twice with PBS 1 ×, pre-incubated in blocking solution (BSA 5 mg/mL and Triton 0.1% in PBS 1 ×) for 15 min, and incubated overnight with anti-Microtubule-associated protein 2 (MAP-2) monoclonal antibody 1:400 (M4403, Sigma-Aldrich) diluted in blocking solution. The day after, cells were washed twice with PBS 1 ×, and incubated for 1 h with a rhodamine-conjugated anti-mouse IgG Cy3 antibody 1:150 (115–165-003, Jackson ImmunoResearch). After two washing with PBS 1 ×, the coverslips were mounted on slides and examined, using the 20 × objective, under a fluorescence microscope (DMRBE, Leica Microsystems), equipped with digital video camera (Spot-RT Slider, Diagnostic Instruments, Mi, USA). The images, acquired in Tiff format, were adjusted for brightness and contrast with the camera software (SPOT Advanced software, v. 4.0.9, Diagnostic Instruments). The specificity of the primary antibody was assessed by analyzing the differential basal expression of MAP-2 protein in undifferentiated and differentiated SH-SY5Y cells (low and high, respectively), and in negative Hep G2 cells (data not shown).

For immunocytochemistry and live staining, the following antibodies were used: rabbit primary antibodies against synaptotagmin 1 for uptake in mouse cells (Oyster550-labelled, 1:100, #105103C3, Synaptic Systems, Göttingen, Germany), synapsin 1,2 (1:1000, #106002, Synaptic Systems), phospho-synapsin 1 (pSyn1S553) (human pS553 corresponds to pS551 in rat; 1:1000, #ab32532, Abcam, Cambridge, UK), VGLUT1 (rat cultures, 1:1000, #135303, Synaptic Systems), VGAT (1:1000, #131003, Synaptic Systems). Antibodies against synapsin 1,2 (1:1000, #106004, Synaptic Systems) were raised in guinea pig. Antibodies against synaptotagmin 1 for monitoring SV recycling in rat neuronal cells (Oyster550-labelled, 1:250, #105311C3, Synaptic Systems and CypHer5E-labelled, 1:200, #105311CpH, Synaptic Systems), against MAP2 (1:1000, #M4403, Sigma-Aldrich, St. Louis, Missouri, USA) and against VGLUT1 (mouse cultures, 1:250 #135311, Synaptic Systems) were raised in mice. Fluorescent secondary antibodies anti-rabbit Alexa 488 (1:1000, #711545152), anti-guinea pig Alexa 488 (1:1000, #706545148), anti-guinea pig Cy3 (1:1000, #706165148), anti-rabbit Cy3 (1:1000 for rat and 1:500 for mouse cultures #711,165,152) and anti-mouse Cy5 (1:1000 #715175150) were all raised in donkey and purchased from Jackson ImmunoResearch Laboratories (West Grove, Pennsylvania, USA).

Cells were rinsed in PBS, fixed with 4% PFA/PBS for 10 min, and permeabilized with 0.25% Triton X-100/PBS for 10 min at room temperature. Samples were blocked with 3.5% BSA/PBS 1 h at room temperature and incubated with primary antibodies overnight at 4 °C. Cells were incubated with appropriate fluorescent secondary antibodies (1:250; Invitrogen) 1 h at room temperature and mounted with hard-setting antifade mounting agent (Aqua-Tex). All antibodies were diluted in 3.5% BSA/PBS. Primary antibodies used were anti-MAP2 (1:2,000, M4403; Sigma), anti-D2R (1:500, AB5084P; Chemicon), anti-IGF-1 (1:200, ab9572; Abcam), and anti-Fos (1:4,000, ABE457; Millipore).