4.4. Serial section electron microscopy

IM and RPE1 cells were fixed with 2.5% glutaraldehyde (G5882; Sigma-Aldrich) in PBS, pH7.4 for 30 min, rinsed with PBS (3 × 5 min), and post-fixed with 2% OsO₄ in dH₂O for 60 min at 4°C. The coverslips were then rinsed in dH₂O, treated with 0.25% tannic acid for 20 min, and stained with 2% uranyl acetate for 60 min. Dehydration was achieved by a series of ethanol solutions (30–50–70–80–96%, 10 min in each solution) followed by acetone (10 min). After dehydration, cells were embedded in Epon 812 and cured for 48 h at 60°C. Serial 70 nm thickness sections were cut with a diamond knife (DiATOME) on a Leica Ultracut UCT ultramicrotome and stained with lead citrate. Images were obtained on a JEOL 1400 microscope operated at 80 kV using a side-mounted 4.0 Megapixel XR401 sCMOS AMT camera (Advanced Microscopy Techniques Corp). Higher-magnification images (40 K) were collected for individual kinetochores. These high-magnification images were subsequently used to trace kinetochores plates. 3-D volumes occupied by the kinetochores were visualized as isosurface models in Amira 5.3.3 (Visage Imaging).