Anti olig2

Cells were washed with PBS and fixed with 4% paraformaldehyde (PFA) for 10 min at room temperature. After fixation, cells were washed three times with PBS. Cells were then blocked at least for 1 h in blocking solution (1% BSA, 3% goat serum, 0.1% Triton in PBS, unless stated otherwise). The blocking solution was removed and the primary antibody (anti-OLIG2 1:400, EMD Millipore, catalog no. 2367; anti-O4 1:200, Immunosolv [product discontinued]; anti-MBP 1:250, Bio-Rad, catalog no. MCA409S; anti-SOX2 1:100, Sigma, catalog no. S9072; anti-SOX9 1:100, EMD Milipore, catalog no. AB5535; anti-GFAP 1:1,000, Sigma, catalog no. G9269; anti-NESTIN 1:10, DSHB Hybridoma, catalog no. rat-401; anti-TUJ1 1:500, BioLegend, catalog no. 801202; anti-A2B5 1:100, Abcam, catalog no. ab53521) was added and incubated at 4°C overnight in blocking solution. After staining with primary antibody, cells were washed three times with PBST for 15 min. Cells were then stained with an appropriate secondary antibody (Alexa Fluor range; 1:1,000 dilutions) for at least 1 h in the dark at room temperature. Cells were washed two times with PBS, stained with DAPI (1:5,000 in PBS) for at least 5 min and washed with PBS twice.

Mice whose samples were employed were fixed with 4% PFA in 0.1 N phosphate buffer (PB) for 24 h at 4°C. They were then washed three times in PB and placed in a 10% sucrose/4% agarose matrix. Sagittal brain sections were obtained on a Leica VT1000 S vibratome (50μm). The sections were subjected to a floating immunolabeling process. For immunofluorescence after performing three washes with PB 0.1 N, the sections were incubated with the following primary antibodies in the blocking solution (1% BSA and 1% Triton X-100 in 0.1 N PB) at 4°C for 24 h: anti-Olig2 (rabbit, 1 : 1000, Millipore); anti-Iba1 (guinea pig, 1 : 1000, Synaptic systems); anti-GFAP (chicken, 1 : 500, Invitrogen); and anti-NeuN (mouse, 1 : 1000, Millipore). Next, five washes were performed with this same blocking solution, and the sections were incubated with the corresponding secondary antibodies conjugated with Alexa fluorophores at 4°C with gentle agitation for 24 h (1 : 1000, Molecular Probes). Finally, three washes were performed with PB and samples were incubated with DAPI diluted 1 : 5000 for 10 min, followed by three additional washes with PB. The sections were placed on slides using FluorSave Images that were taken using a Confocal Spinning Disk SpinSR10 attached to an IX83 inverted microscope (Olympus).