Mouse anti olig2

P2, P16-P21, or 8–10 week-old mice were deeply anesthetized with 0.4 mg/g 2,2,2-tribromoethanol (Avertin), then perfused transcardially with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4 (PB), post-fixed overnight at 4 °C, and cryoprotected in 30% glycerol in 0.1 M PB overnight. 12–40 μm tissue sections were cut on a freezing microtome and processed free-floating. Primary antibodies were diluted in PBS containing 5% goat serum and 0.3% triton-X-100, and incubated with tissue sections for 18 hours at 4 °C in a humidified chamber. Primary antibodies included: chicken-anti-GFP (1:500, Aves or Abcam), mouse anti-NeuN (1:200, Millipore), mouse anti-GFAP (1:500, NeuroMab, clone N206A/8 or Millipore, clone GA5), rabbit anti-SLC7A10, N-term (1:250–1:500, Acris, lot #FGI263), rabbit anti-beta-galactosidase (1:500, MP/Cappel), mouse anti-GLYT2 (1:500, Millipore), mouse anti-OLIG2 (1:500, Millipore), mouse anti-PSD95 (1:2000, NeuroMab), and mouse anti-GPHN (1:200, Synaptic Systems). Secondary detection was conducted at room temperature for 2 hours, using the following antibodies: goat anti-rabbit Alexa Fluor 488, 568, or 647, goat anti-mouse Alexa Fluor 488 or 594, goat anti-chicken Alexa Fluor 488, goat anti-guinea pig Alexa Fluor 594 (1:500, all from Invitrogen). Images were acquired using Zeiss Meta 510, Zeiss Axiovis, or Zeiss 800 and 880 Airyscan microscopes.

Cells from neurosphere differentiation cultures were fixed in 4% PFA with a 30% sucrose solution for 30 min at 37°C. For immunocytochemistry, cultures were preincubated for 1 h in blocking solution (10% goat serum, 0.1% Triton X-100, BSA), followed by overnight incubation with the appropriate primary antibody at 4°C. The following primary antibodies were used: mouse anti-hGFAP (1:500, Sternberger Monoclonal), chicken anti-vimentin (1:200, Millipore), rabbit anti-NG2 (1:200, Millipore), mouse anti-Olig2 (1:200, Millipore), chicken anti-Tuj1 (1:200, Millipore), rabbit anti-active caspase-3 (1:200, Abcam), rabbit anti-HSP27 (1:200, Abcam), and rabbit anti-cathepsin (1:200, Abcam). The corresponding secondary antibodies were incubated for 2 h (Alexa-Fluor 405, 488, 555, or 647 goat anti-mouse, chicken or rabbit; 1:500; Invitrogen), followed by incubation with DAPI (1:1,000, Sigma) for 10 min and rinsing before being mounted on glass slides with Fluorsave (Calbiochem). Analyses were performed with a Nikon 80i fluorescence microscope at 40× or 63× magnification.

Immunofluorescence staining was performed as previously described (Chen et al., 2020 (link)). The primary antibodies included mouse anti-CC1 (1:200, Millipore, Cat: OP80), rabbit anti-NG2 (1:200, Millipore, Cat: MAB5320), mouse anti-Olig2 (1:300, Millipore, Cat: MABN50), rabbit anti-Olig2 (1:300, Millipore, Cat: AB9610), rabbit anti-Sox10 (1:200, Abcam, Cat: ab180862), mouse anti-5mC (1:500, Abcam, Cat: ab10805), and rabbit anti-Ki67 (1:500, Thermo Fisher Scientific, Cat: MA514520). For 5mC staining, before being blocked in 5% BSA, the brain slices were treated with 70% ethanol (pre-cooled on ice) for 5 min and then treated with 1.5 M HCl for 30 min at room temperature.

Brain sections were treated overnight with primary antibody in 5% normal donkey serum/PBS with 0.1% Tween-20 at 4 °C and followed by the appropriate fluorescently conjugated secondary antibody. For samples requiring further unmasking of the epitope, which included anti-Aldh1L1, antigen retrieval was performed by boiling in 6 M sodium citrate buffer at pH 6.0 for 20 min. Slides were then cooled to room temperature and washed 3× with PBS and 0.1% Tween-20, and the standard immunostaining protocol was followed. Well characterized primary antibodies were as follows: rabbit anti-Iba1 (1:500, WAKO), goat anti-Iba1 (Abcam; 1:300), rat anti-SF1 (1:800, kindly provided by Dr. Taro Tachibana, Osaka City University JAPAN), mouse anti-Olig2 (1:300, Millipore), rat anti-LAMP1 (1:800, Millipore), rat anti-CD68 (1:500, BIORAD), goat anti-Sox9 (1:60, R&D systems), rabbit anti-NKX2.1 (1:400, Santa Cruz), sheep anti-Csf1R (1:300, R&D Systems), goat anti-PdgfR alpha (1:150, R&D Systems), rabbit anti-GFAP (1:500; DAKO), rabbit anti-SB100 (1:400; DAKO), and rabbit anti-Aldh1l1 (1:500, Abcam). All appropriate secondary antibodies were Donkey anti-IgG Alexa Fluor conjugated (1:400, ThermoFisher Scientific). All samples were counterstained with Hoechst nuclear stain (1:1000; ThermoFisher Scientific H3570).

At the age of P21, mice (n = 7 in WT, n = 8 in Olig2 cKO) were anesthetized with 1% pentobarbital. Cerebral cortex (anterior cingulated area) and corpus callosum were rapidly removed and frozen. Frozen samples were homogenized and proteins were extracted using RIPA lyses buffer with protease inhibitors (Roche). Lysates containing 40 μg protein were denatured in gel-loading buffer, separated on 10% SDS-PAGE gels, transferred to PVDF membranes and visualized by chemiluminescence (ECL plus, GE Healthcare). Quantification of band intensity was analyzed using Image-Pro Plus software 5.0 (Media Cybernetics). The following primary antibodies were used: mouse anti-Olig2 (1:500; Millipore), mouse anti-MBP (1:1000; Santa Cruz Biotechnology) and mouse anti-β-actin (1:2000; Santa Cruz Biotechnology).

Immunofluorescent labeling on opossum brains from P1 to P25 was performed on slides with brain sections, while at P35, free-floating brain sections were used. Brain sections were incubated for 1 h with either 10% NGS, 10% normal chicken serum (Sigma–Aldrich), or 1% bovine serum albumin in PBS. After, the sections were incubated overnight in rabbit anti-calbindin (1:1,000, Chemicon), rabbit anti-NeuN (1:50, Cell Signaling), mouse anti-Olig2 (1:100, Millipore, Kankakee, IL, USA), rabbit anti-Tbr1 (1:200, Abcam), rabbit anti-GFAP (1:500, Dako), rabbit anti-TrkC (1:500, Cell Signaling), and rabbit anti-NT3 (1:300, Alomone Labs) primary antibodies. The appropriate secondary antibodies, namely, goat anti-rabbit 568, chicken anti-goat 488, and goat anti-mouse 488 were used. Finally, the sections were mounted on slides and cover-slipped with a 60% glycerol solution in PBS.