Epon araldite

After the treatment with the Slab51 supernatant for 24h, the morphological and ultrastructural alterations of G. duodenalis trophozoites were investigated by transmission electronic microscopy (TEM). To this aim, trophozoite samples were fixed in phosphate-buffered 0.1M of 2% glutaraldehyde (pH 7.4), post-fixed in phosphate-buffered 1% OsO4 and, after dehydration, embedded in Epon/Araldite (Polyscience Inc., Warrington, PA, USA). Ultrathin sections (70 nm) were placed on 200-mesh nickel grids supplied with formvar-carbon film (Agar Scientific Ltd, Stansted, UK). Grids were then stained with lead citrate and uranyl acetate and examined with a JEOL 1200-EX transmission electron microscope (JEOL, Peabody, MA, USA).

Dissections were fixed in 4% paraformaldehyde and 2.5% glutaraldehyde in cacodylate buffer, post-fixed in 1% osmium tetroxide, and stained in uranyl acetate before embedding in Epon-Araldite (Polysciences, Warrington, PA, USA). Lead stained 0.1 μm sections were examined on a JEOL 1200EXII microscope at 80 kV. We examined over 80 sections from 4 specimens of each genotype.

Transmission electron microscopy (TEM) was used to analyze morphological characteristics of autophagy. U251 and C6 cells were seeded in incubation bottles for 24 h. Then cells were treated with MOX for 48 h, the untreated cells served as control group. Then the cells were harvested, washed and fixed overnight with 2.5% glutaraldehyde containing 1% tannic acid at 4°C. After washing, the cells were dehydrated using a graded series of ethanol solutions, 10 min at a time, transferred to propylene oxide and embedded in epon araldite (Polysciences, Inc., Polybed 812, USA). The ultrathin sections were observed using a JEM-100CX transmission electron microscope (Shanghai Yongming Automatic Equipments Co. Ltd., H-7650, China) and representative images were photographed and analyzed.