Smi 99p 100

Immunocytochemistry was performed as described^{34 (link)}. Primary antibodies were used at a dilution of 1:500. Antibodies used in this study are: Rat anti-MBP (Millipore MAB386), rabbit anti-Olig2 (Millipore AB9610), mouse anti-MBP (Covance SMI-99P-100), Rabbit anti-Neurofilament H (Millipore AB1989). After a 1 hour incubation at room temperature with appropriate secondary antibodies, cells were counterstained with DAPI (Invitrogen R37606). Cells were stained with Alexa Fluor 488-phalloidin (Invitrogen A12379) during secondary antibody incubation to visualize F-actin. Cells were mounted on glass slides and imaged on a Zeiss LSM700 confocal microscope with 20x objective or on a Keyence BZ-X710 epifluorescence microscope with 20x objective for tiling (9×9).

Tissue samples were prepared, run on SDS-PAGE gels and transferred to PVDF membranes as described^{34 (link)}. Transferred membranes were incubated with primary antibodies overnight at 4°C. Primary antibodies used were as follows: Rabbit anti-Olig2 (1:2,000 Millipore AB9610), Mouse anti-MBP (1:2,000 Covance SMI-99P-100), Mouse anti-MOG (1:3,000 Millipore MAB5680), Rabbit anti-Neurofilament H (1:10,000 Millipore AB1989), Rabbit anti-GDE2 (1:1000), Rabbit anti-PDGF receptor α (1:2,000 Cell Signaling Technology 3174), Mouse anti-Actin (1:10,000 Millipore MAB1501). After 1 hour incubation at room temperature with appropriate HRP-conjugated secondary antibodies, membranes were developed by film or by using a digital imaging system (KwikQuant, Kindle Biosciences).

Cultured cells were fixed with 4% PFA solution for 10 minutes and permeabilized in PBS with 0.3% Tween-20 for 10 minutes followed by blocking with PBS containing 1% bovine serum albumin (BSA) and 0.15% Tween-20 for 1 hour at room temperature. The cells were incubated with primary antibodies diluted in PBS (1:500) overnight at 4°C. Primary antibodies used were as follows: Rat anti-MBP (Millipore MAB386), rabbit anti-Olig2 (Millipore AB9610), guinea pig anti-Olig2 (from B. Novitch), mouse anti-MBP (Covance SMI-99P-100), mouse anti-beta-Tubulin III (Sigma-Aldrich T8578), Rabbit anti-Neurofilament H (Millipore AB1989), rabbit anti-GFAP (Agilent Z0334), mouse anti-Active-β-catenin (Anti-ABC) (Millipore 05–665). After incubation with appropriate secondary antibodies (1 hour room temperature), cells were counterstained with DAPI (Invitrogen R37606). To visualize F-actin network, cells were stained with Alexa Fluor 488-phalloidin (Invitrogen A12379) during secondary antibody incubation. Cells were mounted on slides with mounting reagent and imaged using confocal microscope (Zeiss LSM700) with 20x objective and on epifluorescence microscope (Keyence BZ-X710) with 10x objective for tiling (3×3).