Pa1 16789

Organoids were fixed in 4% PFA for 30 min, infiltrated with sucrose, and embedded in Optimal Cutting-Temperature (OCT) media (Fisher Scientific, Waltham, MA, USA) for cryo-sectioning. Seven-micron thick sections were taken and blocked in 1%BSA/PBS-0.3% Triton X100 for 1 h at room temperature. Organoid sections were incubated either with PSD95 (ab12093, abcam, Waltham, MA, USA), NG2 (ab275024, abcam), Olig2 (ab136253, abcam), or Abca1 (PA1 16789, Thermo Fisher) antibodies diluted 1:250 in blocking solution at 4 °C overnight. Slides were washed and incubated for three hours at room temperature in appropriate secondary antibody (A11055, Invitrogen; ab6718, abcam) diluted 1:500 in blocking solution. Slides were washed twice for 5 min in PBS, incubated for 5 min in 1:1000 DAPI, washed twice more with PBS, and cover slipped. Slides were imaged using a confocal microscope (Zeiss LSM 880 confocal microscope, Carl Zeiss AG, Oberkochen, Germany).

The cultured cells and tissues were lysed by RIPA buffer (Beyotime) mixed with 0.1 mmol/L phenylmethylsulfonyl fluoride. The concentration of total proteins in the extracts was quantified using a BCA protein assay kit. Then, proteins were subjected to SDS-PAGE, followed by immunoblotting with rabbit polyclonal antibody against CTRP12 (PA5-46452, 1:500, ThermoFisher), rabbit polyclonal antibody against ABCA1 (PA1-16789, 1:800, ThermoFisher), rabbit polyclonal antibody against ABCG1 (GTX30598, 1:500, GeneTex, Irvine, CA, USA), rabbit polyclonal antibody against CD36 (PA1-16813, 1:1000, ThermoFisher), mouse monoclonal antibody against SR-A (sc-166184, 1:500, Santa Cruz, TX, USA), rabbit polyclonal antibody against LXRα (L5044, 1:1000, Sigma-Aldrich) and rabbit monoclonal antibody against β-actin (ab115777, 1:1000, Abcam, Cambridge, MA, USA). After a series of rinses with PBS-T, the membranes were further incubated with HRP-labeled secondary antibodies (1:5000, Beyotime). The protein bands were visualized using Tanon 5500 (Shanghai, China) and BeyoECL Plus (Beyotime). The densitometry values were determined using Image J software and normalized to β-actin values.

Total protein extracts were prepared as described [17 (link)]. Equal amounts of protein were resolved by SDS-PAGE and analyzed with anti-LXRα (1 : 1,000, ab135039, Abcam, Cambridge, MA), anti-TNF-α (1 : 1,000, ab183896, Abcam), anti-interferon-γ (IFN-γ; 1 : 1,000, EPR1108, Abcam), anti-ABCA1 (PA1-16789, Thermo scientific, Fremont, CA), and anticytoskeletal actin (1 : 10,000, A300-491A, Bethyl Laboratories, Montgomery, TX) antibodies. The secondary antibody used with each was goat anti-mouse antibody (1 : 2,000, AbFrontier, Seoul, Korea), except for anticytoskeletal actin (1 : 20,000). Following transfer and blotting, the proteins of interest were visualized by enhanced chemiluminescence (Pierce, Rockford, IL) and analyzed.