Fei tecnai biotwin transmission electron microscope

Transmission electron microscopy was performed as previous study (Hwang et al., 2021b (link)). Washed epididymal sperm were pelleted by centrifugation and fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1M cacodylate buffer pH7.0 for 30 minutes at RT and for 1 hour at 4 °C. The fixed pellets were briefly washed with 0.1M cacodylate buffer pH7.0 and placed in 2% agar. The chilled blocks were trimmed and rinsed with 0.1M cacodylate buffer followed by placed in 0.1% tannic acid in 0.1M cacodylate buffer for 1 hour. The samples were washed and post-fixed in 1% osmium tetroxide and 0.15% potassium ferrocyanide in 0.1M cacodylate buffer for 1 hour. Post-fixed samples were rinsed with the cacodylate buffer and distilled water and subjected to en bloc staining in 2% aqueous uranyl acetate for 1 hour. The samples were dehydrated with ethanol series and infiltrated with epoxy resin Embed 812 (Electron Microscopy Scienes). The resins were placed in silicone molds and backed for 24 hours at 60 °C. The blocks were sectioned in 60 nm depth using Leica UltraCut UC7 and the sections were collected on the formvar/carbon-coated grids. Sections on the grids were stained using 2% uranyl acetate and lead citrate. The stained grids were imaged using MORADA CCD camera (Olympus) equipped in FEI Tecnai Biotwin Transmission Electron Microscope (FEI, Hillsboro, OR) at 80 KV.