Mabn50

Sections were permeabilized for 1 hour in a 4% donkey serum/PBS blocking buffer containing 0.3% Triton X-100 (MilliporeSigma) and incubated overnight at 4°C with the following primary antibodies diluted in a 1% donkey serum/PBS buffer: mouse anti-TH (Merck, MAB318, 1:2,000), goat anti-HA (Genscript, A00168, 1:500), mouse anti-Olig2 (Merck, MABN50, 1:500), mouse anti-Flag (1:1,000, MilliporeSigma, F3165, 1:500), rat anti–Lamp-2 (Abcam, Abl93, ab25339, 1:1,000), and rabbit anti-LC3 (Novus Biological, NB2220, 1:1,000). Following incubation with primary antibodies, tissues were washed with PBS 3 times for 10 minutes and incubated for 1.5 hours at room temperature with a combination of corresponding donkey anti-species IgG conjugated to AlexaFluor probe (Invitrogen, 1:400). Tissues were then washed with PBS and mounted in DAPI-containing mounting media (Vectashield). Illustrative images were acquired using a confocal microscope Leica SP8 at the BioImaging Center in Bordeaux.

Immunocytochemistry was performed to characterize NSs in vitro with anti-Nestin antibodies (Abs) (a marker for NSCs, 19483-1-AP, ProteinTech), glial fibrillary acidic protein (GFAP) (a marker for astrocytes, 60190-1-Ig, ProteinTech), βIII-tubulin (a marker for immature neurons, 11224-1-AP, ProteinTech), and Olig2 (a marker for immature oligodendrocytes, MABN50, MERCK). The nuclei in the NSs were treated with 4′-6-diamidino-2- phenylindole (DAPI) (Sigma-Aldrich, USA) counterstaining. PE- and FITC-conjugated secondary Abs (Jackson Laboratories, West Grove, PA, USA) were added, followed by imaging of NSs with confocal microscopy (Leica sp8, Germany).

Tissue sections or adherent cultured cells were incubated for one hour at room temperature in blocking solution (50 mmol/L Tris-HCl, pH 8.0, 0.1 mol/L NaCl, 0.1% Triton X-100, 3% NGS, 0.1% BSA) prior to overnight primary antibody incubation (4°C). Primary antibodies for immunofluorescence studies were: SOX2 (1:1000 dilution, Ab5603, Millipore), Ki67 (1:500 dilution, ab15580, Abcam), BrdU (1:500 dilution, ab6326, Abcam), NeuN (1:500 dilution, ab177487, Abcam), TUJ1 (1:200 dilution, T8660, Sigma-Aldrich), GFAP (1:500 dilution, Z0334, Agilent), MBP (1:300 dilution, SMI-99P, Covance), OLIG2 (1:200 dilution, AB9610, Millipore), OLIG2 (1:50 dilution, MABN50, Millipore), IBA1 (1:200 dilution, ab5076, Abcam), CD31 (1:100 dilution, ab24590, Abcam), ACTA2 (1:400 dilution, ab5694, Abcam), GFP (1:2000 dilution, ab13970, Abcam), CD31 (1:300 dilution, AF3628-SP, R&D), CD146 (1:100 dilution, 134701, BioLegend). We used immunofluorescence staining with Alexa Fluor 488, 555, or 647 (Life Technologies) and biotin-streptavidin-Alexa Fluor-conjugated secondary antibodies (Jackson ImmunoResearch), as well as horseradish peroxidase-based Vectastain ABC Kit (Vector Laboratories). Image acquisitions were performed using a Zeiss LSM880 confocal microscope and image editing done using ZEN, Photoshop or ImageJ.

The following antibodies were used for immunohistochemistry or western blot analyses: rabbit anti-Nsun5 (15449-1-AP, Proteintech Group Inc., Wuhan, China; Western blot,1:300; IF,1:100), rat anti-MBP (MAB386, Millipore, Billerica, MA, USA; Western blot,1:500; IF,1:600), rabbit anti-PDGFRα (ab203491, Abcam, Cambridge, UK, 1:500), mouse anti-BrdU (MAB4072, Millipore, 1:1000), rabbit anti-NG2 (AB5320, Millipore, 1:200), rabbit anti-BLBP (ab32423, Abcam, 1:1000), mouse anti-CC1 (OP80, Millipore, 1:200), rabbit anti-Ki67 (ab16667, Abcam, 1:500), mouse anti-olig2 (MABN50, Millipore, 1:300), rat anti-NG2 (MAB6689-SP, Millipore, 1:400), rabbit anti-CDK2 (ab32147, Abcam, 1:5000), rabbit anti-CDK1 (19532-1-AP, Proteintech, 1:1000), rabbit anti-cleaved caspase-3 (ab2302, Abcam; 1:300) and rabbit anti-RhoA (10749-1-AP, Proteintech, 1:1000). The secondary antibodies-donkey anti-mouse or anti-rat-conjugated to either Alexa Fluor 488 (Jackson ImmunoResearch, West Grove, PA, USA, 1:500) or 555 (Jackson ImmunoResearch; 1:500) and the biotinylated goat anti-mouse secondary antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:200) was directed against the primary IgG antibody species.

Mice were perfused with 4% paraformaldehyde and post-fixed overnight in the same solution at 4 °C. Tissue samples were then transferred to 70% ethanol, sequentially dehydrated and embedded in paraffin. Four-micrometer sections were cut, deparaffinized and rehydrated. Antigen retrieval was performed by incubating slides in sub-boiling (94 °C) citrate buffer (pH 6.0) for 15 min. Slides were incubated in blocking buffer (20% Normal Goat Serum / 1% BSA / PBS 1X) for 1 h at room temperature and then incubated overnight at 4 °C with the primary antibodies in 1% BSA / PBS 1X. Antibodies used are: mouse anti-OLIG2 (Millipore, MABN50, 1:200), mouse anti-NeuN (Millipore, MAB377, 1:200), mouse anti-GFAP (Sternberger Inc., SMI-22, 1:200), rabbit anti-BIN1 (Abcam, ab185950, 1:200), rabbit anti-GOT2 (Abcam, ab171739, 1:200). After rinsing with Tris-buffer / 2% milk, sections were processed with he appropriate Alexa Fluor conjugated secondary antibodies (1:1000 in 1% BSA / PBS 1X, Invitrogen), washed with Tris-buffer, and mounted using Fluoromount-G with DAPI. All images were acquired using a Zeiss Observer A1 fluorescent microscope or Zeiss LSM780 upright Confocal. Quantification was carried out on two-three sections per mouse and three-four mice for each genotype evaluated using ImageJ.