Tecnai g2 tf20 super twin

Samples were fixed with 1% (w/v) glutaraldehyde in PBS for 10 min, then loaded on a carbon/formvar-coated grid (5 µl each sample) and dried under vacuum for 20 min. Samples were incubated with 2% uranyl acetate (10 µl) for 30 s, then dried under vacuum for 20 min. The ultrastructure of each sample was observed by a transmission electron microscopy (FEG-TEM, FEI Tecnai G2 TF20 Super TWIN).

For immunofluorescence confocal microscopy, three-dimensional structured illumination microscopy (3D-SIM), and electron microscopy, cells were processed as previously described^{32 (link)}. Briefly, cells on coverslips were treated with aphidicolin for synchronization at the G1/S phase transition and released with fresh medium at the indicated times. The cells were fixed in methanol and incubated with the indicated primary antibodies. After wash, the cells were incubated with Alexa Fluor 488-, Alexa Fluor 568-, or Alexa Fluor 647-conjugated secondary antibodies (Invitrogen). In some experiments, cells were immunostained with the Alexa Fluor 568-conjugated CEP120 antibody. The samples were viewed on a confocal system (LSM 700 or LSM880 Airyscan systems; Carl Zeiss). The 3D-SIM super-resolution images were acquired using a Zeiss ELYRA system equipped with a Plan Apochromat 63 × /1.4NA oil-immersion objective and the ZEN software (Carl Zeiss). For electron microscopy (EM), cells were grown on Aclar film (Electron Microscopy Sciences), fixed, and embedded in Spurr’s resin as previously described^{32 (link)}. Thin sections (100 nm) were stained with 4% uranyl acetate and Reynold’s lead citrate and the samples were examined with an electron microscope (T FEG-TEM; FEI Tecnai G2 TF20 Super TWIN)^{32 (link)}.

Thin sections placed on nickel grids were blocked with 5% BSA in PBS for 20 min and incubated with the mouse anti-ATP5A (500x, ab14748, Abcam) antibodies in incubation buffer (1% BSA in PBS) for 2 h. Grids were subsequently washed with incubation buffer three times (10 min each). Secondary antibodies, goat anti-mouse IgG (EM.GMHL15, BB International) with 15 nm gold conjugations at 20-fold dilution were further applied and incubated for 1 h. After washing with PBS, the immune-complexes were fixed with 1% glutaraldehyde in PBS for 5 min and washed three times with distilled water (10 min each). The specimens were inspected by TEM operating at 120 kV (FEI Tecnai G2 TF20 Super TWIN).