Clone o4

The following primary antibodies were used in the present immunohistochemical experiments: mouse monoclonal IgG against nestin (clone Rat-401; dilution 1:100; Santa Cruz Biotechnology, Santa Cruz, CA, USA); rabbit polyclonal IgG against, green fluorescent protein (GFP: A6455, Molecular Probes; dilution 1:1000), Pax6 (PD022, Medical and Biological Laboratories; dilution 1:1000); guinea pig IgG against GFAP (YN-2011; dilution 1:400)^{51 (link)}; goat polyclonal antibody against Sox2 (sc-17320, SantaCruz; dilution 1:2,000); rat IgG against BrdU (Abcam, Cambridge, UK, AB6326; dilution 1:1,000); chicken polyclonal antibody against vimentin (AB5733, Chemicon; dilution 1:12,000). For nuclear staining, sections were incubated with DAPI (1 μg/ml; Dojindo, Kumamoto, Japan).
The following primary antibodies were used in the present immunocytochemical experiments: mouse monoclonal IgG against NeuN (clone A60, Chemicon, Merck-Millipore, Temecula, CA; dilution 1:300); mouse monoclonal IgM against O4 (clone #O4, R&D Systems, McKinley Place, MN; dilution 1:1,000); guinea pig IgG against GFAP (YN-2011; dilution 1:400)^{51 (link)} and MAP2 (NH-2004; dilution 1:500)^{52 (link)}.

OPCs were isolated from the neonatal (P7–P9) mouse brain to perform cell culture studies, whereas pro-OLs were isolated from adult brains (6–8 weeks old mice) to assess the efficiency of Cre-mediated recombination. Mouse pups were anesthetized on ice and sacrificed by decapitation according to institutional guidelines. Adult mice were anesthetized and perfused intracardially with ice cold Hanks’ balanced salt solution (HBSS) without Ca²⁺/Mg²⁺ to remove blood from the vasculature. Brains were extracted and cerebral cortices dissected and diced into small pieces, which were then incubated in a mixture of papain (0.9 mg/mL), cysteine (0.2 mg/mL) and DNase I (39 U/ml) at 37 °C for 20 min on an orbital shaker. The digestion was stopped by adding a trypsin inhibitor with a final concentration of 5 mg/mL, and the tissue was dissociated into a single-cell suspension by gentle mechanical trituration. OPCs and pro-OLs were isolated by immunopanning at room temperature using antibodies directed against PDGFRα (also known as CD140a; 1:300 dilution, clone APA5, BD Biosciences, #558774) and O4 (1:300, clone O4, R&D Systems, MAB1326) cell surface markers, respectively, following the methods of Emery and Dugas^{101 (link)}.

To assess the efficiency of Cre-mediated recombination in astrocytes, astrocytes were isolated from the brain of adult mice using the semi-automated gentleMACS™ Octo Dissociator with Heaters, the Adult Brain Dissociation Kit and the Anti-ACSA-2 MicroBead Kit, according to the standardized methods and protocols developed by Miltenyi Biotec. Astrocytes were further enriched by cell sorting using the APC-conjugated anti-mouse ACSA-2 (1:50, clone REA969, Miltenyi Biotec, 130-116-245) and Alexa 488-conjugated mouse anti-Oligodendrocyte Marker O4 (1:50, clone O4, R&D Systems, FAB1326G) antibodies, according to the gating strategy described in Supplementary Fig. 8.