M3821

Thirty µm-thick serial frozen sections were stained with 0. 1% cresyl violet to visualize tissue morphology. For immunohistochemistry, sections were washed in 0.1 M phosphate buffer saline (PBS), blocked in 10% goat serum and 3% albumin bovine serum for 2 hr, and incubated with primary antibodies overnight at 4°C. Signal was detected with Alexa fluor 546 or 488 fluorescent secondary antibodies (1∶1000, Invitrogen). Primary antibodies were: goat anti-choline acetyl transferase (ChAT, 1∶500, AB144p, Millipore), rat anti-CD11b (1∶1000, MCA711G, AbD serotec), rabbit anti-GAP-43 (1∶2000, AB5312, Chemicon), mouse anti-NeuN (1∶1000, MAB377, Chemicon), rabbit anti-MBP (1∶200, M3821, Sigma), rabbit anti-c-Jun (1∶500, Cat. No. 9165, CST) and mouse anti-Parvalbumin (PV, 1∶2000, Cat. No. MAB1572, Millpore).

Cells were homogenized in 100 μL Kaplan buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.4), 1% NP-40, 10% glycerol, and a protease inhibitor cocktail (Roche Diagnostics)). The lysate was subject to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and WB analysis with a standard procedure [62 (link)] using antibodies against AKT (1:1000; CST 4691; Cell Signaling Technology, Beverly, MA, USA), phospho-AKT (1:1000; CST 4056; Cell Signaling Technology), ERK1/2 (1:1000; CST 4695; Cell Signaling Technology), phospho-ERK1/2 (1:1000; CST 9101; Cell Signaling Technology), p38 (1:1000; CST 9212; Cell Signaling Technology), phospho-p38 (1:1000; CST 9215; Cell Signaling Technology), JNK (1:1000; CST 9252; Cell Signaling Technology), phospho-JNK (1:1000; CST 4668; Cell Signaling Technology), c-JUN (1:1000; CST 9165; Cell Signaling Technology), phospho-c-JUN (1:1000; CST 3270; Cell Signaling Technology), MBP (1:1000; M3821; Sigma-Aldrich), MAG (1:1000; MAB1567; Chemicon, Temecula, CA, USA), P0 (1:1000; ab31851; Abcam, Cambridge, UK), HIF1α (1:1000; CST 14179; Cell Signaling Technology), and β-actin (1:1000; CST 4970; Cell Signaling Technology). Protein expression levels were determined using the MF-ChemiBIS 3.2 imaging system (Berthold Technologies, Bad Wildbad, Germany).

Flow cytometry was performed with an LSR Fortessa (BD Biosciences) for data acquisition and fluorochrome-conjugated antibodies targeting CD133 (Miltenyi Biotec, AC133, 1:20), CD15 (BD Biosciences, 1:100), CD44 (in-house Hermes 3, 1:500), CD49f (Jomar Biosciences, 14-0495, 1:100), EGFR (in-house, M528, 0.5 μg/ml), EGFRvIII (in-house, 806, 0.5 μg/ml), PDGFRα (R&D Systems, MAB1264, 0.5 μg/ml), EphA2 (in-house, 1F7, 0.5 μg/ml), EphA3 (in-house, IIIA4, 0.5 μg/ml), EphA4 (in-house, 1F9, 0.5 μg/ml), FGFR1 (Abcam, ab823, 1:100), FGFR2 (Abcam, ab89476, 1:100), FGFR3 (R&D Systems, MAB766, 0.5 μg/ml), c-Met (R&D Systems, MAB3582, 0.5 μg/ml), Sox2 (R&D Systems, MAB2018, 0.5 μg/ml), Nestin (R&D Systems, MAB1259, 0.5 μg/ml), GFAP (Dako, Z0334, 1:100), β-III tubulin (Promega, G7121, 1:100) and MBP (Sigma Aldrich, M3821, 1:100). Cells were incubated with antibody on ice for 30 minutes in PBS containing 2% FBS. Where required, cells were fixed and permeabilised using Intracellular Fixation & Permeabilization Buffer (eBioscience) and stained with antibodies in 1 × Permeabilization Buffer. Data analysis was performed using Flowjo (Treestar) software.