Epoxy resin

Medium was first decanted before cells were fixed with 2.5% glutaraldehyde (Sinopharm Chemical Reagent Co., Ltd.) at 4˚C for 15 min. Cells were subsequently collected by centrifugation at 362 x g and 37˚C and stored at 4˚C. Cells were rinsed three times with 0.1 M phosphate buffer (Sinopharm Chemical Reagent Co., Ltd.) for 15 min, followed by dehydration in a 30, 50, 70, 80, 85, 90 and 100% ethanol gradient for 15-20 min in each alcohol solution. The penetrant was composed of epoxy resin (Sinopharm Chemical Reagent Co., Ltd.) and acetone (Sinopharm Chemical Reagent Co., Ltd.). The infiltrated sample was placed in an embedding plate, before addition of embedding epoxy resin. The samples were embedded with epoxy resin (Hubei Xinkang Pharmaceutical Chemical Co., Ltd.) in a 60˚C incubator for 48 h. An ultramicrotome was used to slice the sections to 80-100 nm thickness. Uranyl acetate was added to the sections and incubated at room temperature for 15 min. The sections were dried overnight at room temperature and observed under an electron microscope.

Baclofen tablets (positive reference drug) were purchased from Fuan Pharmaceutical (Group) Co., Ltd (Ningbo, China). Paraformaldehyde (4%) was obtained from Biosharp Co., Ltd (Hefei, China). Choral hydrate was purchased from Tianjin Kemiou Chemical Reagent Co., Ltd (Tianjin, China). Nissl staining solution, in which the active ingredient is cresyl violet, was purchased from Beyotime Company (Shanghai, China). Glutaraldehyde, osmium acid, acetone, epoxy resin, uranium acetate, and lead citrate were purchased from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China). Anti-BDNF antibody, anti-TrkB antibody, and anti-KCC2 antibody were obtained from Abcam (Cambridge, UK). Horseradish enzyme labeled chain affinity and diaminobenzidine were purchased from ZSGB-BIO Co., Ltd (Beijing, China). The inverted fluorescence microscope (TH4-200) was obtained from Olympus Corporation (Tokyo, Japan).