V900933

Cells were plated and cultured in poly-D-lysine-coated, glass-bottom, 96-well microtiter plates (ViewPlate microplate (#6005530, PerkinElmer). Plasmids expressing target membrane proteins were transfected into cells using Lipofectamine 3000 (#L3000015; Invitrogen). 24 h after transfection, cells were fixed by 4% paraformaldehyde for 20 min at room temperature, followed by 3 washes with PBS (pH 7.4) for 5 min each. Cells were then blocked with 1% BSA (#V900933, Sigma; dissolved in PBS) for 30 min at 37 °C. After blocking, cells were incubated with corresponding antibodies (diluted in 1% BSA/PBS, 2 μg mL⁻¹) at 4 °C overnight. 2 μg mL⁻¹ secondary antibody, Alexa FluorTM 488 goat anti-human IgG (H + L) (1:1000 dilution; #A11013, Invitrogen), was then mixed with the cells in a 1% BSA/PBS buffer at room temperature for 1 h. DAPI (#10236276001, Roche) was added for nuclei staining. After 3 times washing with PBS, the stained cells were kept in PBS for immunofluorescence analysis on a confocal microscopy (ZEISS LSM710).

Approximately 50,000 SCs were seeded in the purified DRG neurons, and cocultured with DMEM-HG medium containing 10% FBS, 0.4% glucose (15023021, Gibco), 2 mM l-glutamine and 50 ng/ml NGF. After 3 days of co-cultivation, basal layer formation was initiated for approximately 4 days with DMEM medium supplemented with 0.2% bovine serum albumin (BSA; V900933, Sigma), ITS (I3146, Sigma) and 50 ng/ml NGF, and then the medium were changed to DMEM medium containing 50 μg/ml l-ascorbic acid (A0278, Sigma), 50 ng/ml NGF and 15% FBS to induce myelination for 2–3 weeks until myelin sheath being achieved, with fresh media provided every 2 days. The myelin associated glycoprotein (MAG) immunostaining was performed to assess myelination in the co-culture system.

For tissue IF, the antigens were extracted as described in the IHC assay. Tissue sections were blocked with Tris-buffered saline-Tween 20 (TBST) containing 5% bovine serum albumin (BSA) (Sigma-Aldrich, V900933) for 1 h at room temperature (RT). Subsequently, the tissue sections were blocked in TBST containing 5% BSA for 1 h at RT. After blocking, the samples were incubated overnight at 4 °C with primary antibodies followed by incubation with Alexa Fluor-conjugated secondary antibodies (Invitrogen) for 1 h at RT. Nuclei were counterstained using 4′,6-diamidino-2-phenylindole (DAPI, Servicebio, G1012). For cellular IF, cells were seeded into confocal cell culture dishes and fixed using 4 % formaldehyde, permeabilized with 0.2% Triton X-100, and blocked with 5% BSA for 1 h at RT. Subsequently, the samples were incubated with primary antibodies at 4 °C overnight and then with secondary antibodies at RT for 1h. Finally, the nuclei were stained with DAPI. Images were captured using a laser confocal microscope (LSM780, Zeiss). Details of all antibodies are given in Supplementary Table 2.

Cells were plated on glass coverslips in 6-well plates, washed with prewarmed 1×PBS three times, and fixed in 4% paraformaldehyde (VWR BT140770) in PBS for 10 min at room temperature (RT). After washing, the cells were permeabilized in 0.2% Triton X-100 (Sigma Aldrich, T8787), 1% BSA (Sigma, V900933) for 15 min at RT. After a wash with 1×PBS, the cells were incubated with 2% BSA at RT for at least 40 min and subsequently incubated with primary antibodies (anti-CTCF, Millipore 07–729/Active motif 61311/Abcam ab128873, 1:800 dilution) in 1% BSA overnight at 4°C. After three washes with 1×PBS, 1% BSA, 0.1% Tween 20 for 10 min, the cells were incubated with Alexa Fluor-tagged secondary antibody (donkey anti-rabbit IgG, Alexa Fluor 488, Invitrogen, A-21206, at a 1:1000 dilution) in the dark for 1 h at room temperature. Then, the cells were washed three times, mounted with a fluorescent mounting solution with DAPI (ZSGB-BIO, ZLI-9557) and Vectashield (Vector Laboratories, H-1000), sealed with colorless nail polish, and imaged on a NIKON A1RSi + confocal microscope with a 100×/1.45 oil objective using NIS-Elements software (Nikon, USA).