Ultrastructural Analysis of MNT-1 Cells

MNT-1 cells cultured on carbonated sapphire disks or CryoCapsules (CryoCapCell; Heiligenstein et al., 2014 (link)) were transfected with control, Myo6, or WASH siRNAs or treated for 60 min with 25 µM DMSO or TIP. Cell were high-pressure frozen with HPM100 (Leica Microsystems) or HPM Live μ (CryoCapCell) and then freeze substituted in anhydrous acetone containing 1% OsO4/2% H₂O for 64 h in a freeze substitution system (AFS; Leica Microsystems). Cells were embedded in epon 812 (TAAB Laboratories Equipment) and processed for sectioning and contrasting with uranyl acetate and lead citrate. Ultrathin sections were observed at 80 kV transmission electron microscope (Tecnai Spirit G2; Thermo Fisher Scientific) equipped with a QUEMESA CCD camera (EMSIS) using iTEM software (EMSIS). For ultrathin cryosections and immunogold labeling, MNT-1 cells were grown on six-well plates and fixed with 2% PFA/0.2% glutaraldehyde/0.1 M phosphate buffer. Cells pellets were embedded in 10% gelatin and infused in 2.3 M sucrose. Gelatin blocks were frozen and processed for ultracryomicrotomy. Ultrathin sections (90 nm) were double-immunogold labeled using PAG 10 or 15 nm and analyzed by EM as described above.