Ab9610

For histological examination of brains, tissue was fixed in 4% formaldehyde for at least 12 h. The tissue was dehydrated, embedded in paraffin, and sectioned at 2 μm according to standard protocols. Hematoxylin and eosin (HE) stainings were applied according to standard protocols. 3,3’-Diaminobenzidine (DAB) stainings were performed on a Ventana Benchmark system using the ultraView or OptiView DAB detection kit (all Roche Diagnostics, Basel, CH). The following antibodies were used: Cleaved Caspase-3 (CC-3): Cell Signaling #9664, RRID:AB_2070042 (1:100); GFP: Abcam #ab290, RRID:AB_303395 (1:500); Ki67: Abcam #ab15580, RRID:AB_443209 (1:100); MYC: Zeta Corporation #Z2734RL (1:25); Nestin: Abcam #ab221660, RRID:AB_2909415 (1:2000); NeuN: Merck #MAB377, RRID:AB_2298772 (1:50); OLIG2: Merck #AB9610, RRID:AB_570666 (1:200); SMARCA4: Abcam #ab110641, RRID:AB_10861578 (1:25); and SOX2: Abcam #92,494, RRID:AB_10585428 (1:200).

CNPase Atlas AMAb91072 (1:2000), MBP Serotec MCA409S (1:250), PECAM1 BD Pharmingen 550274 (1:100), S100ß Thermo MA5-12969 (1:100), IBA1 Abcam ab178846 (1:500), Laminin Abcam ab11575 (1:300), Olig2 Millipore AB9610 (1:100), CC1 Abcam ab16794 (1:300).

WM was dissected from 300 µm-thick brain sections obtained from Nx and Hx mice at P18, and digested in Hanks’ Balanced Salt Solution (Gibco, 14170-161) containing papain (13 units/ml, Sigma, T4762), DNAse (5 units/ml, Sigma, D5427) and trypsin (Sigma, T4799) for 30 min at 37 °C. Cells were dissociated by trituration and resuspended in Hanks’ Buffer containing 1 M HEPES (BioSource, P305), 15% sucrose, and Penicilin/Streptomycin. Cells were then plated onto poly-L-lysine-coated dishes at a density of 10 cells/µl and cultured for 10 days in D-MEM/F12 medium (Gibco, 11330-032), supplemented with 1% N2 and 1% B27 (Gibco, 17502-048, 17504-044), and with 20 ng/ml EGF and 10 ng/ml bFGF (Upstate Biotechnology, 01-407, 01-114). To immunolabel differentiated cells, standard protocols were used^{7 (link),16 (link)} with primary antibodies against GalC (1:200, Galactocerebroside; Abcam ab142) and Olig2 (1:200, Abcam, ab9610).

The following primary antibodies (antigen [clone], dilution, vendor, cat. no.) were used for these studies: NPC1 [EPR5209], 1:500, Abcam, ab134113; NeuN [A60], 1:500, MilliporeSigma, MAB377; Ki67 [polyclonal], 1:200, Abcam, ab15580; Vinculin [hVIN-1], 1:2000, MilliporeSigma, V9131; Neurofilament [N52], 1:500, MilliporeSigma, MAB5266; H3K27me3 [C36B11], 1:200 [IF], 1:1000 [WB], Cell Signaling Technologies, 9733 S; H3K9me3 [D4W1U], 1:200 [IF], 1:1000 [WB], Cell Signaling Technologies, 13969 S; Histone H3 [96C10], 1:2000, Cell Signaling Technologies, 3638 S; MBP^{12 (link)}, 1:100 [IF], 1:500 [WB], Abcam, ab7349; OLIG2 [polyclonal], 1:500 [IF], 1:1000 [WB], MilliporeSigma, AB9610; SOX10 [SP267], 1:400, Abcam, ab227680; SOX10 [A-2], 1:200, Santa Cruz Biotechnology, sc-365692; H3K27me3 [polyclonal], ActiveMotif, cat. #39155; H3K27ac [polyclonal], ActiveMotif, cat. #39133.

Cells were fixed in 4% paraformaldehyde, permeabilised and blocked in 0.1% bovine serum albumin plus 3% goat serum solution. Samples were incubated overnight with primary antibodies followed by incubation with appropriate secondary antibodies (1:1000; Invitrogen) and 4′,6-diamidino-2-phenylindole (DAPI). Immunopositive cells were quantified using ∼5000 cells (minimum of ten random fields). Total cell number was determined by DAPI nuclear staining. The following primary antibodies were used: mouse nestin (1:10; Developmental Studies Hybridoma Bank, Rat-401), human nestin (1:500; R&D Systems, MAB1259), mouse Sox2 (1:100; Abcam, 92494), human Sox2 (1:50; R&D Systems, MAB2018), BLBP (also known as Fabp7) (1:200; Santa Cruz, sc-30088), GFAP (1:1000; Sigma, G3893), Tuj1 (also known as Tubb3) (1:1000; Biolegend, 801202), GFP (1:1000; Abcam, AB13970), Olig2 (1:400; Millipore, AB9610), mCherry (1:500; Abcam, AB167453), V5 tag (1:1000; eBioscience, 14679682), p53 (1:400; Cell Signaling, 2524).

Immunostaining was performed as described elsewhere [32 (link),44 (link)]. Cells were fixed with pre-warmed 4% (w/v) paraformaldehyde (PFA), 0.15% glutaraldehyde, and 0.1% (w/v) Triton X-100 dissolved in phosphate buffered saline (PBS) for 15 min at 37°C. After washing with PBS twice, cells were treated with 0.1% (w/v) sodium borohydride (Honeywell Fluka, 71321, Charlotte, North Carolina, United States) dissolved in PBS for 20 min at RT. Primary antibodies were diluted in blocking solution (3% (w/v) bovine serum albumin (BSA) and 0.1% (w/v) Triton X-100 in PBS). The following primary antibodies were used: anti-Olig2 antibodies (Merck Millipore, AB9610), anti-MBP antibodies (Abcam, ab7349), anti-tubulin antibodies (Sigma-Aldrich, T6199). Alexa fluor conjugates (Thermo Fisher Scientific, A11007, A28175 and A21244) were used as secondary antibodies. Nuclei were stained with Hoechst (Molecular Probes, 33342). All images were acquired with an inverted light microscope (Axio Observer Z1, Carl Zeiss MicroImaging, Inc., Jena, Germany) equipped with epifluorescence optics. Images were captured with a CCD camera controlled by ZEN software (Carl Zeiss MicroImaging, Inc.). Areas stained for MBP and axon length were drawn or traced manually by using draw spline contour and curve graphic elements of the ZEN software (Carl Zeiss Micro Imaging, Inc.).

Coronal brain sections (40 μm in thickness) through the entire brain and spinal cord were prepared in serial order and processed for histological analysis as previously described [63 (link)]. For quantification of oligodendrocyte lineage cells, immunostaining was performed with the following primary antibodies: anti-Olig2 (Millipore, Rabbit, AB9610), anti-CC1 (Abcam, Mouse, ab16794), and anti-MCM2 (BD, Mouse, 610701). 4′,6-diamidino-2-phenylindole (DAPI; Sigma) was used for counterstaining. For quantification of motor cortex neuron and cerebellar Purkinje neuron density, immunostaining was performed with the following primary antibodies: anti-NeuN (Millipore, Mouse, MAB377), and anti-Calbindin (Swant, Rabbit, CB-38a). Images were acquired on a Zeiss LSM 780 single-photon confocal system using a multi-track configuration. For counting cells in the corpus callosum, images were acquired to include only the corpus callosum at the midline. In the spinal cord, images were acquired to include only the white matter from serial sections of lumbar spinal cord. Stereological quantification of immunostained cells was carried out using NIH ImageJ [64 (link)].