Tuj 1

Proteins from P19 cells were homogenized in M-PER lysis buffer (Pierce Chemical Co., Rockford, IL). Extracted proteins (15 µg) were separated by SDS-polyacrylamide gel electrophoresis and were electrophoretically transferred onto a membrane according to the previously described approach [20] (link). The membrane was blocked in blocking buffer and incubated with antibodies to NELL2 (Santa Cruz Biotech., Santa Cruz, CA, Catalogue No. sc-54637), β-actin (Sigma-Aldrich, Catalogue No. A5441), Tuj1 (Santa Cruz Biotech., Catalogue No. sc-5274), NeuN (Millipore, Billerica, MA, Catalogue No. MAB377), N-cadherin (Abcam, Boston, MA, Catalogue No. ab76057), phosphorylated ERK (pERK) (CELL Signaling Technology, Beverly, MA, Catalogue No. 9101), ERK (Santa Cruz Biotech., Catalogue No. sc-153), or c-Fos (Santa Cruz Biotech., Catalogue No. sc-7202). Blots were developed using horseradish peroxidase-conjugated anti-goat secondary antibody (Santa Cruz Biotech., Catalogue No. sc-2020), anti-mouse secondary antibody (Santa Cruz Biotech., Catalogue No. sc-2005) or anti-rabbit secondary antibody (Santa Cruz Biotech., Catalogue No. sc-2004). Immunoreactivity was detected with an enhanced chemiluminescence (ECL) kit (Amersham Pharmacia Biotech., Buckinghamshire, UK).

The procedure had been previously described (Chen et al., 2011 (link)). In brief, the cells were lysed and the protein concentrations of the lysates were determined. Then, the lysates were separated by 8% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked, followed by incubation with primary antibodies and then POD-labeled secondary antibodies (1:12500; Roche, Mannheim, Germany). The primary antibodies were used as follows: TuJ1 (1:1000), TET1 (1:500), srGAP3 (1:1000), and α-Tubulin (Santa Cruz; 1:5000). The signals were detected by BM Chemiluminescence Western Blotting kit (Roche, Mannheim, Germany).

ReNcell VM cells were proliferated in chamber slices coated with laminin and subsequently differentiated as described above. After the cells had been incubated for 30 min with blocking solution (5% normal goat serum with 0.3% TritonX-100 in PBS), the mouse primary antibody Tuj-1 (Santa Cruz) was added and incubated for 30 min at room temperature. After a wash with PBS, the secondary antibodies Alexa Fluor 488 or Alexa Fluor 568 goat anti-mouse (Molecular Probes) were added. Incubation was for 60 min at room temperature. Subsequently, the slides were embedded with mounting medium containing DAPI (Vectashield) and the staining was imaged using a fluorescent microscope system (BZ-8000; Keyence Deutschland GmbH).

Brain tissues were sectioned on a standard rotary microtome (Leica, Berlin, Germany). Tissue sections were collected and counterstained with hematoxylin-eosin (HE). Then, the sections were subjected to staining with the primary antibodies. Primary antibodies were tyrosine hydroxylase (TH, 1:1000) (Santa Cruz Biotech., Dallas, TX, USA), P-CREB (1:500, Upstate Comp., Lake Placid, NY, USA), caspase 3 (1:500), or MDA (1:100). After raining with PBS, sections were stained with 3,3′-diaminobenzidine (DAB) as described [24 (link)]. Subsequently, the selected sections were further performed by using Tuj1 (1:500), GFAP (1:500), and IBA1 antibody (1:200, Santa Cruz Biotech., Dallas, TX, USA). The stained sections were counterstained with DAPI, mounted and examined microscopically.