Anti olig2 antibody

Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich). After 15–20 minutes, we washed cells with 0.1% BSA/PBS washing buffer 3 times and blocked for 1 hour by adding 0.3% triton X-100 (Sigma-Aldrich) and 10% normal goat serum (Invitrogen). After blocking, cells were incubated with primary antibodies at 4°C overnight. Anti-NG2 antibody (1: 200; Chemicon, Temecula, CA, USA), anti-Nestin antibody (1:500; BD Biosciences, NJ, USA), anti-Olig2 antibody (1:1000; Chemicon), anti-MBP antibody (1:1000; Abcam, Cambridge, UK), anti-A2B5 antibody (1:200; R&D systems), anti-PDGF-R antibody (1:200; R&D systems), and anti-O4 antibody (1:500; R&D systems) were used. Biotin was reacted for 30 minutes, and fluorescence-tagged antibody was reacted for one hour. Cells were mounted in VECTASHIELD with DAPI mounting medium (Vector Laboratories, Burlingame, CA, USA).

Cells were fixed in 4% paraformaldehyde (Sigma-Aldrich) for 15∼20 minutes at room temperature. Cells were blocked and permeabilized with 10% normal goat serum (Pel-Freez) and 0.3% Triton X-100 (Sigma-Aldrich) in 0.1% bovine serum albumin/phosphate buffered saline (BSA/PBS) for 1 hour. Samples were incubated with primary antibodies at 4℃ overnight. The samples were then washed three times with 0.1% BSA/PBS and incubated with secondary antibodies at room temperature for 1 hour. The cells were counterstained with 4’,6-diamidino-2-phenylin-dole (Vector Laboratories) and mounted using a mounting solution. Images were captured using an epifluorescence microscope (LEICA Microsystems). The primary antibodies used in this study were as follows: anti-NESTIN (1：500; BioLegend), anti-SOX2 (1：1,000; Sigma-Aldrich), anti-ZO1 (1：50; Thermo Fisher Scientific), anti-PAX6 (1：200; Novus Biologicals), anti-TUJ1 (1：2,000; BioLegend), anti-TH (1：2,000; Pel-Freez), anti-glial fibrillary acidic protein (GFAP, 1：2,000; DAKO), anti-OLIG2 antibody (1：1,000; Chemicon), anti-major histocompatibility complex (MHC) Class I+HLA A/B (1：200, MHC-I; Abcam), anti-MHC Class II (1：200, MHC-II; Abcam). The secondary antibodies used were Alexa Fluor 488 (1：500; Ther-mo Fisher Scientific), Rhodamine, or Cy3 (1：500; Jackson ImmunoResearch Laboratories).

Immunohistochemical analysis was performed using primary antibodies for glial fibrillary acidic protein (GFAP), ionized calcium-binding adaptor molecule 1 (Iba-1), myelin basic protein
(MBP), neurofilament 200 kDa (NF), and oligodendrocyte transcription factor (olig-2). After de-paraffinization, sections were transferred into citric acid buffer (pH 6.0) and boiled for 20
min at 98°C in a microwave processor (MI-77, Azuma, Tokyo, Japan) to retrieve the antigens. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide for 15 min and then the
sections were pre-incubated with 10% normal goat serum for 30 min at room temperature. Next, the sections were incubated with a mouse monoclonal anti-GFAP antibody (diluted 1:400, Dako
Denmark, Glostrup, Denmark), a rabbit polyclonal anti-Iba-1 antibody (diluted 1:2,000, Wako, Osaka, Japan), a mouse monoclonal anti-MBP antibody (diluted 1:1,000, Merck, Darmstadt, Germany),
a mouse monoclonal anti-neurofilament antibody (diluted 1:500, Millipore, Burlington, MA, USA), or a rabbit polyclonal anti-olig-2 antibody (diluted 1:500, Millipore) at 4°C overnight. The
reaction was revealed using 3, 3′-diaminobenzidine tetra-hydrochloride as a chromogen (Agilent, Tokyo, Japan), and counterstaining was performed with hematoxylin.