Rabbit anti olig2

The antibodies used were as follows: mouse anti-β-catenin antibody, mouse anti-Stat3 antibody and mouse anti-BrdU antibody were from BD Biosciences; mouse anti-Bmi-1 antibody and rabbit anti-OLIG2 and rabbit anti-POU3F2 was from Abcam; mouse anti-PTEN antibody and rabbit anti-CHOP antibody were from Cell Signaling Technology; mouse anti-Nestin antibody and rabbit anti-Sox2 antibody were from Millipore, mouse monoclonal anti-CD133 (W6B3C1 clone) was from Miltenyi Biotec.

To examine the histopathological changes in the striatum, 10 weeks (14 weeks after birth) after AAV-DJ-82Q injection, the mice (n = 7 per group) were deeply anesthetized with diethyl ether and then intracardially perfused with saline and cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB, pH 7.4). Brains were removed and post-fixed for 24 h and cryoprotected in 10, 20 and 30% sucrose in PBS serially at 4°C. Serial coronal slices (30-μm thickness) of striatum were acquired on a model CM3050S freezing microtome (Leica, Wetzlar, Germany) and collected in sequence as free-floating sections on PBS. The sections from the mid-coronal level of the striatum (Franklin and Paxinos, 2008 ) were processed for immunofluorescence stain as previously described (Lee et al., 2016a (link)). Briefly, the brain sections from each group (n = 3 per brain) were incubated with mouse anti-HTT protein (1:400; catalog MAB5374, clone EM48; Millipore), rabbit anti-NeuN (1:5,000; Abcam), rabbit anti-cleaved caspase-3 (1:500; Cell Signaling Technology), rabbit anti-Iba-1 (1:2,000; WAKO), mouse anti-GFAP (1:5,000; Millipore) and rabbit anti-Olig2 (1:500; Abcam) overnight with gentle agitation at room temperature. The sections were followed with a mixture of secondary antibodies and examined with confocal imaging system (LSM five PASCAL; Carl Zeiss, Germany).

For flow cytometric analysis of EB-NPCs, cells were collected using Accutase and stained with mouse anti-Nestin, mouse anti-human Sox1, and mouse anti-Sox2 per manufacturer’s instructions using the Human Neural Lineage Analysis kit (BD). For immunofluorescence microscopy, EB-NPCs were seeded on Geltrex-coated slides, fixed with 4% paraformaldehyde, and stained with rat anti-Nestin (1:500; Millipore), rabbit anti-Sox2 (1:200; Epitomics), or rabbit anti-Pax 6 (1:50; BioLegend) before addition of respective secondary antibodies (goat anti-rabbit AlexaFluor 568 and goat anti-rat AlexaFluor 488; both from Thermo Fisher). For analysis of multipotency, EB-NPCs were cultured in neuronal differentiation medium (Neurobasal, 1X B27, 1X GlutaMAX), astrocyte differentiation medium (DMEM [Thermo Fisher], 1X N2, 1X GlutaMAX, 1% FBS [Atlanta Biologicals]), or oligodendrocyte differentiation medium (Neurobasal, 1X B27, GlutaMAX, 30 ng/ml T3 [Sigma]) for at least 14 days before being fixed with 4% paraformaldehyde and stained with mouse anti-beta-III-tubulin (1:500; Abcam), rabbit anti-GFAP (1:200; Thermo Fisher), rabbit anti-NG2 (1:200; Chemicon), or rabbit anti-Olig2 (1:100; Abcam). Slides were cover-slipped and mounted using VectaShield Hard Set Mounting Medium with DAPI (Vector Labs), and all images were captured using a Nikon Eclipse Ti inverted microscope.