Brain sample collection for immunohistochemistry

Mice were perfused via the cardiovascular system with PBS followed by 4% paraformaldehyde (Electron Microscopy Sciences, Hatfield, PA). Brains were removed and post-fixed in 4% paraformaldehyde 1 day at 4°C. The brains were kept in PBS with 0.02% sodium azide at 4°C until sectioning. For sectioning, the brains were embedded in 4% UltraPure low melting point agarose (Thermo Fisher Scientific, Waltham, MA) and were coronally sectioned by vibratome (VT1000S; Leica Microsystems, Wetzlar, Germany) at a thickness of 50 µm. Brain sections of 50 µm were collected and stained every 0.15 mm. The brain sections were stored as free-floating in PBS with 0.02% sodium azide at 4°C until staining.

The free-floating sections were incubated with primary antibody in blocking solution (10% horse serum, 0.3% triton X-100, and 0.02% sodium azide in PBS) overnight at room temperature. The next day, sections were incubated with fluorescence-conjugated secondary antibody for 1.5–2 hours at room temperature. Between each step and after secondary antibody staining, sections were thoroughly washed with PBS or PBS with 0.1% triton-X-100 at least three times for 10 minutes each. The stained free-floating sections were then mounted onto the Superfrost Plus microscope slides (Fisher Scientific, Hampton, NH) in PBS. Excess PBS from adhered sections were carefully removed. Slides were dried at room temperature for 2–5 minutes. 150–200 µl of ProLong Diamond, anti-fade mountant with DAPI (Thermo Fisher Scientific, Waltham, MA) was applied to the slides before placing the coverslip. The slides were left to set overnight before imaging.

Primary antibodies used for imaging throughout and their dilutions were: mouse anti-NeuN (1:1000; MAB377; Millipore Sigma, Burlington, MA), goat anti-Olig2 (1:500; AF2418; R&D Systems, Minneapolis, MN); mouse anti-CC1 (1:250; NB600–1021; Novus Biologicals, Littleton, CO); rabbit anti-NG2 (1:300; AB5320; Millipore Sigma, Burlington, MA); chicken anti-MBP (1:250; CH22112; Neuromics, Edina, MN); mouse anti-neurofilament (1:250; 837802; Biolegend, San Diego, CA), rabbit anti-PLP (ab183493, Abcam, Cambridge, UK). The fluorescent-conjugated secondary antibodies were donkey anti-goat (1:1000; A-32814, A-21082, A11057; ThermoFisher Scientific, Waltham, MA), donkey anti-rabbit (1:1000; A-21206, A-10042, A-31573; ThermoFisher Scientific, Waltham, MA), and donkey anti-mouse (1:1000; A-21202, A-10037, A-31571; ThermoFisher Scientific, Waltham, MA), and donkey anti-chicken (1:1000; A-11041; A-11039; A-21449, Thermo Fisher Scientific, Waltham, MA).