Sox10

Tissues were fixed in 10% formalin (VWR, Radnor, PA, 48218–700) for 24 hours at room temperature, then stored in 70% ethanol until embedding in paraffin. For immunohistochemistry, antigen retrieval was performed at 97 °C for 20 minutes using pH 6.0 citrate buffer composed of 10 mM sodium citrate and 0.05% Tween 20. The following antibodies were used for immunohistochemistry: Sox10 (Santa Cruz Biotechnology, Dallas, TX, sc-17342, 1:100), PHGDH (Sigma-Aldrich, HPA021241, 1:2000), Ki67 (BD Biosciences, Franklin Lakes, NJ, 550609, 1:40), and cleaved caspase 3 (Cell Signaling Technology, Danvers, MA 9661S, 1:300). Slides were scanned using an Aperio slide scanner (Leica Biosystems, Wetzlar, Germany), and images were analyzed using Aperio ImageScope. Ki67 and PHGDH staining was quantitated using the Positive Pixel Counter v9 in Aperio ImageScope. For Ki67, a region of each tumor measuring 1.5 mm by 0.7 mm was quantitated at 10x magnification. For PHGDH, 5 mammary glands were quantitated per mouse at 20x magnification using a color threshold of 0.1. cleaved caspase 3 staining was quantitated by scoring 300 cells per tumor as positive or negative. Samples were de-identified before all staining quantitation.

The sciatic nerves of mice at defined ages were dissected and fixed for 15 min in 4% PFA in 0.1 M sodium phosphate buffer (pH 7.4), embedded in optimal cutting temperature (OCT) compound, cryoprotected in 25% sucrose, and sectioned at 12 μm as longitudinal or cross-sections using a cryostat or at 30 μm using a vibratome. Tissue sections or cells were permeabilized and blocked in blocking buffer (0.3% Triton X-100 and 5% normal donkey serum in PBS) for 1 h at 25 °C, followed by incubation with primary antibodies overnight at 4 °C. Antibodies against the following proteins were used: HDAC3 (rabbit; Santa Cruz Biotechnology, sc-11417), MPZ (rabbit; Abcam, ab31851), MBP (goat; Santa Cruz Biotechnology, sc-13914 and mouse; BioLegend, 836501), NF-M (rabbit; Millipore, AB1987), SOX10 (goat; Santa Cruz Biotechnology, sc-17342), EGR2 (rabbit; Santa Cruz, sc-20690), Ki67 (rabbit; Thermo Scientific, RM-9106), SOX2 (goat; Santa Cruz Biotechnology, sc-17320), HDAC5 (mouse; Sigma, H4538), CD31 (rat; BD Pharmingen, 553370), and CASPR (mouse; NeuroMab, 75-001). Secondary antibodies conjugated to Cy2, Cy3, or Cy5 were from Jackson ImmunoResearch Laboratories (catalog numbers 705-165-147, 705-225-147, 711-225-152, 711-165-152, 711-175-152, 715-165-150, and 712-165-150). All images were acquired using a Nikon C2+ confocal microscope.

Mouse E12.5 spinal cords were dissected, dissociated, and processed for ChIP assays. The samples were pre-cleared with protein G beads and immunoprecipitated using NFIA antibody (abcam), Sox10 (rabbit-polyclonal), or control IgG (Santa Cruz). The DNA was purified and PCR was preformed using region specific primers. See supplemental information for primer sequences. HEK293 or HEK293T cell lines cells were transfected with pGL3-reporter constructs and a CMV-β-galactosidase vector using Superfect transfection reagent (Qiagen). Cells were harvested and analyzed for luciferase activity; β-galactosidase was used to normalize for transfection efficiency.
Co-immunoprecipitation was performed by transfecting P19 or Oli-Neu cells with Flag-NFIA and/or HA-Sox10; harvested cell lysates were subject to immunoprecipitation using a specific antibody or IgG control and protein G agarose beads. See supplemental information for additional information.

Slides were thawed to room temperature, and lines drawn to partition the edges of sections on the glass slides using a PAPpen (ImmEdge Pen; Vector Labs H4000). The sections were rehydrated, blocked, permeabilized and immunostained with primary antibodies: PAX6 (BioLegend, 1:200), SOX2 (Santa Cruz, 1:100), β-tubulin III (Sigma, 1:1000), FOXG1 (Takara, 1:500), SOX10 (Santa Cruz, 1:100), Ki67 (Thermo Fisher, 1:200), Nestin (Millipore, 1:200), GFAP (Millipore, 1:400), S100beta (DAKO Potts, 1:400), CTIP2 (Abcam, 1:500), SATB2 (Abcam, 1:50), MAP2AB (Abcam, 1:1000-2000), NeuN (Millipore, 1:200), TBR1(Abcam, 1:500), BRN2 (Millipore, 1:500), MEF2C (Novis, 1:200), AT8 (Invitrogen, 1:1000), PHF1 (gift from Peter Davies,1:1000), Total Tau (DAKO Potts, 1:200), DA9 (gift from Peter Davies, 1:1000), ELAVL4 (Santa Cruz, 1:200), TIA1 (Santa Cruz, 1:100), G3BP1 (Protein Tech, 1:200), VGLUT1 (gift from Susan Morton, Tom Jessell, 1:16,000), Calbindin1 (Swant, 1:10,000), Homer1 (SYSY, 1:250) and Synapsin1 (Millipore, 1:100). Primary antibodies were incubated overnight at 4C, washed three times with PBS and then incubated with corresponding Alexa Fluor conjugated secondary antibody (1:333-1000) for 1 hour at room temperature. Sections were coverslipped and imaged using fluorescence and confocal microscopy (Zeiss AXIO Observer.Z1; Zeiss 780).