Glutaraldehyde

Each group of aorta samples was dissected into pieces and fixed with 2.5% glutaraldehyde (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) at 4 °C. After stimulation, HUVECs were washed with PBS and harvested by trypsinization, centrifuged at 1000 rpm for 5 min and fixed with 2.5% glutaraldehyde at 4 °C for 24 h. Then the samples were post-fixed with 1% osmium tetroxide at 4 °C (1 h for HUVECs, 2 h for aorta tissue) and washed with PBS three times. Specimens were dehydrated in ethanol (Beijing Chemical Works, Beijing, China) with a gradient series and 100% acetone (Beijing Chemical Works), infused with Epon812 (Serva, New York, NY, USA) and embedded in pure Epon812 at 65 °C (48 h for HUVECs, 72 h for aorta tissue). After the semi-thin sections were observed, the aortic cross-section was located, and five serial ultra-thin sections (70 nm) from each rat were stained with 4% uranyl acetate (Kojima Chemicals Co., Ltd., Tokyo, Japan) and lead citrate (Alfa Aesar, Ward Hill, MA, USA). The samples were examined with TEM (H-7650, Hitachi, Tokyo, Japan).

The EAE mice were euthanized at 21 dpi. The spinal cords were first fixed with 2.5% glutaraldehyde (Sinopharm Chemical Reagent Co., Ltd) in PBS overnight, then washed three times by PBS and postfixed with 1% OsO4 (SPI-CHEM) in PBS for 1.5 h and then rinsed by PBS. After, the samples were first dehydrated in ascending dilution series of ethanol for 15 min at each step, and then dehydrated for 20 min by alcohol. Next, the samples were transferred to absolute acetone (Sinopharm Chemical Reagent Co., Ltd) for 20 min. The samples were then placed in 1:1 (1 h) and 1:3 (3 h) mixture of absolute acetone and the final Spurr resin mixture (SPI-CHEM) at room temperature, and then transferred to the final Spurr resin mixture overnight. After that, samples were embedded in Spurr resin and heated at 70 °C for more than 9 h, and then were sectioned in LEICA EM UC7 ultratome. Sections were stained by uranyl acetate and alkaline lead citrate (Sinopharm Chemical Reagent Co., Ltd) for 5–10 min, respectively, and observed in Hitachi Model H-7650 TEM. The analysis was performed with Image J. The G-ratios of myelinated fibers were calculated as the ratio of the axonal diameter to the myelinated fiber diameter as measured from different locations as described previously [50 (link)].

Silkworm cocoons (Bombyx mori, 185 Nd-s) were obtained from Sericultural Research Institute, China Academy of Agricultural Sciences (Zhenjiang, Jiangsu, China). LF 20/40 alginate (high molecular weight, HMW) was obtained from FMC Biopolymer (Philadelphia, PA, USA). Low molecular weight (LMW) alginate was generated from HMW alginate using gamma irradiation at 50 KGy for 4 hours with a cobalt-60 source. Glutaraldehyde and CaCl₂ were purchased from Sinopharm Chemical Reagent Co., Ltd (China). C57BL/6J mice were supplied by Department of Experimental Animals, Tongji Medical College, Huazhong University of Science & Technology (Wuhan, China). The mouse myoblast cell line (C2C12) was purchased from the American Type Culture Collection (Rockville, MD, USA).