Tr 100 bg

The myelin staining followed the instructions of kit (Biosensis, TR-100-BG). Briefly, the sectioned samples were air-dried at 37°C for 2 h on a slide warmer until thoroughly dry. Then they were washed in distilled water for 2 minutes, and incubated in preheated solution A for about ~ 12 minutes in a clean covered Coplin jar in water bath, 60–65°C. The slides were rinsed in distilled water for 2 minutes and incubated in 1 X Solution B for 3 minutes. Following that, the slides were rinsed in distilled water for three times, each 5 minutes. The samples were then dehydrated via nature air-drying and immersed in xylene for 1–2 minutes to total remove water. Finally, the samples were mounted using a non-aqueous mounting media DPX (Sigma, 06522–100ML) and imaged by a lightscope.

Transverse brain sections of 12-μm thickness were prepared and HEK293 cells were seeded on LAB-TEK chamber slides (Thermo Fisher Scientific, 154453) for immunoflorescence assays as previously described (18 (link), 42 (link)). The following antibodies were used: anti-RNF220 (1:200; Sigma-Aldrich, HPA027578), anti-PDGFRα (1:400; R&D, AF1026), anti-CC1 (1:200; Millipore, OP80), anti-Sox10 (1:300; Oasis Biofarm, OB-PGP001), anti-BrdU (1:200; Bio-Rad, MCA6144), anti-Ki67 (1:200; Abcam, ab15580), and fluorescence-conjugated secondary antibodies, Alexa Fluor 488/555/594 donkey/goat anti-rabbit/mouse/rat/ guinea pig immunoglobulin G (1:400; Invitrogen; A11076, A11055, A21202, A48269, A21206, A31572). Images were captured using a light microscope (Olympus, VS120) and analyzed with ImageJsoftware.
For myelination analysis, 20-μm brain coronal sections were prepared with a cryostat microtome (Leica, CM1850UV) and stained with the Black-Gold II kit (Biosensis, TR-100-BG) according to the manufacturers’ instructions. Images were captured using a light microscope (Olympus, VS120) and analyzed with ImageJ software.