Myelin was isolated as previously described66. In brief, myelin was isolated after mechanical homogenization of perfused brains and washed twice with PBS containing 0.32 M sucrose. Afterward, the CNS homogenate was carefully transferred on top of a 0.85 M sucrose solution in PBS. The CNS sucrose gradient was centrifuged at 3200×g for 60 min. The interphase containing myelin was isolated and subjected to osmotic shock by washing it twice in distilled water. The protein amount of purified myelin was assessed using a Pierce™ BCA Protein Assay Kit, and 1 mg of myelin was stained with pHrodo dye (all from Thermo Fischer Scientific) in PBS pH 8.2, according to the manufacturer’s instructions, followed by washing in PBS pH 7.4 to remove excess dye.
