D8417

Cells were seeded onto a 12-well plate attached with crawling slides (φ 20 mm). After the treatment, 4% paraformaldehyde was used to fix cells for 15 min, and washed with PBS three times, and incubated with 0.5% Triton-X for 15 min at RT. Slides were then incubated with 5% BSA at 37 °C for 1 h blocking. Primary antibodies (targeting the proteins of interest) were used to incubate with cells containing 1% BSA and 0.3% Triton X-100 (T9284, Sigma-Aldrich, St. Louis, MO, USA) at 4 °C overnight. FITC-labeled secondary antibody was used to stain the cells. After being washed three times with PBS, DAPI (1 μg/mL, D8417, Sigma-Aldrich, St. Louis, MO, USA) was used to detect nuclei for 5 min at RT. The images were captured using DMi8 Microsystems BmbH (Leica, Wetzlar, Germany).

After pretreatment with citrate buffer and blocking solution (see above) for 1 h at RT, three free-floating sections per animal (n = 6 per genotype and age) were stained with the following primary antibodies over-night at 4 °C: OLIG2 (1:500, AB9610, Merck Millipore), platelet-derived growth factor receptor alpha (PDGFRα, 1:250, AF1062, R&D Systems), α-syn (1:200, 15G7, ALX-804-258, Enzo Life Sciences), phosphorylated α-syn at Ser129 (pS129-α-syn, 1:500, ab51253, Abcam), and TPPP/p25α (1:200, ab92305, Abcam). For fluorescence staining, the following secondary antibodies were incubated for 1 h at RT: Alexa Fluor 647 donkey anti-rabbit (1:1000, 711-605-152, Dianova), Alexa Fluor 568 donkey anti-goat (1:1000, A11057, Life Technologies), and Alexa Fluor 488 donkey anti-rat (1:1000, A21208, Life Technologies). Nuclei were counterstained with DAPI (1:10,000, D8417, Sigma) for 10 min. To analyze the cell density in the ROI, three Z stack images were taken in each region using the fluorescence Observer microscope (Zeiss) in conjunction with the ZEN blue software. Cell density and the area of OLIG2⁺ cells were quantified using the cell counter plugin and the analyze particles plugin of the ImageJ software, respectively.

After the formation of SC spheroids in AggreWell^TM400 for 24 h, they were collected, dissociated into single cells using Accutase^TM (Millipore, SCR005), suspended in the mTeSR^TM1 medium with 10 μM ROCK-inhibitor Y-27632, and seeded on laminin-521 (LN521, Biolamina) coated black 96-well μ-plates (ibidi, 89626) to form a 2D cell monolayer. LN521 coating was prepared by incubating 10 μg/ml LN521 diluted in 1× DPBS with Ca⁺ and Mg⁺ either overnight at 4°C (slow coating) or for 2 h at 37°C (fast coating). After cells attached for 3 h, they were fixed in 4% paraformaldehyde for 10 min, permeabilized with 0.1% Triton X-100 for 10 min, and thereafter blocked with 10% normal goat or donkey serum (Millipore, Burlington, MA, United States) for 1 h. Cells were incubated with primary antibodies overnight at 4°C and then with secondary antibodies conjugated with Alexa Fluor 594 or Alexa Fluor 488 (Invitrogen) for 1 h at room temperature. Cell nuclei were stained with DAPI (Sigma-Aldrich, D8417, 12.5 μg/ml in MilliQ water) for 2 min. Primary and secondary antibodies used for immunostaining in this study are listed in Supplementary Table 2.

OPCs were seeded at a density of 40,000 cells per well in 96-well plates coated with in poly-L-ornithine (Sigma, P3655) and laminin (Sigma, L2020) in differentiation permissive media supplemented with 40 ng/mL T3 (Sigma, T6397). Cells were treated with TDCIPP (Sigma, 32951), TMPP (Santa Cruz, sc-296611), or TBPP (Millipore, 34188) at a final concentration of 20uM. DMSO (Sigma, D2650), was added at 1:1000 to negative control wells. As described preivously^{50 (link)}, cells were live stained with anti-O4 (1:100, CCF Hybridoma core), anti-O1 (1:100, CCF Hybridoma core), and fixed with 4% Paraformaldehyde (Electron Microscopy Sciences, HP1–100Kit) after 1-, 2-, and 3-days post-plating. Cells were then stained overnight with anti-MBP (1:4000, Abcam, ab7349) followed by staining with DAPI (1 μg/mL, Sigma, D8417). The Operetta High Content Imaging and Analysis system was used to image 4 fields per well and the percentage of O4, O1, and MBP-positive cells was quantified using the number of DAPI-positive live cells per field.

For immunofluorescence, tissues were deparaffinized by heating and rehydrated with xylene and ethanol. Antigen retrieval was performed with citrate antigen retrieval buffer, pH 6.0, and tissues were permeabilized with 0.5% triton, quenched with glycine, and blocked with 5% donkey serum. Sections were then incubated with anti-acetaminophen (Bio-Rad, 0016-0104) and anti-CYP2E1 (Abcam, ab28146) primary antibodies overnight followed by incubation with secondary antibodies (Alexa Fluor Anti-Rabbit Donkey 594 and Alexa Fluor Anti-Sheep 647) for 1 hour. Finally, nuclear counterstaining was performed by incubating slides with DAPI (Sigma-Aldrich, D8417). Staining was visualized using the Olympus IX83 microscope and Olympus DP80 camera using Olympus cellSens software.