Goat anti olig2

Mice brains were cut into several brain slices (25 µm) by microtome. At room temperature, the brain slides were sealed with 1% immune tissue blocking fluid for 2 h and incubated overnight with rabbit anti-MBP (1:100, Abcam, Cambridge, UK), anti-Iba-1 (1:100, Wako, Japan), anti-CC1 (1:100, Millipore, Burlington, MA, USA), goat anti-Olig2 (1:100, Millipore, Burlington, MA, USA), anti-PDGFRα (1:100, Millipore, Burlington, MA, USA) respectively at 4 °C. Subsequently, the brain slides were incubated with the corresponding secondary antibody at room temperature for 1 h. Then, the corpus callosum site of slices was photographed with a multiphoton laser scanning confocal microscopy system (Olympus Fluoview FV1000) or common fluorescence microscope (Leica DMI6000).

To determine which cells captured the EB dye, coronal brain slices from the SE-24hEB group were washed with TBS (0.02 M Tris-buffered saline, pH 7.4), and blocked with 5% skimmed milk powder and 0.3% Triton X-100 diluted in TBS for 2 h at room temperature (RT). Slices were incubated overnight at 4°C with primary antibodies diluted in TBS containing 5% skimmed milk powder and 0.1% Triton X-100. The antibodies used were: mouse anti-NeuN (1:150; #MAB377–Millipore) for neuron detection, rabbit anti-GFAP (1:150; #G9269–Sigma Aldrich) for astrocyte labeling, goat anti-OLIG-2 (1:150; #AB9610–Millipore) for oligodendrocyte lineage, and goat anti-IBA-1 (1:150; #AB5076–Abcam) for microglia. Slices were then washed with TBS and incubated with fluorophore-labeled secondary antibody (1:200, Alexa Fluor 488; Thermo Fisher Scientific) in TBS with 0.l% Triton X-100 for 2 h at RT, in the dark. Slides were mounted as described above and were examined and photographed with the same fluorescence microscope mentioned. Evans blue dye was observed in red (546 nm emission filter), immunostained cells in green (488 nm excitation filter), and nuclear DAPI in blue (UV filter).