Tecnai 20 tem

HT22 cells were cultured in 35 mm glass-bottomed culture dishes (NEST Biotechnology Co., Wuxi, China) to 50%–60% confluency. The next day, 10 mM L-serine was treated at the 5 h before rotenone treatment. 1 μM Rotenone was treated for 19 hours. Then, cells were fixed in 2.5% glutaraldehyde-mixed 2% paraformaldehyde solution for 1 h followed by post-fixation in 2% osmium tetroxide for 1 h at 4 °C. The block was stained in 2% uranyl acetate O/N. Subsequently, the cells were dehydrated in an ethanol series and then embedded in Poly/Bed 812 resin (EMS, Hatfield, PA, USA). Embedded samples were sectioned (70 nm) with an ultra-microtome (Leica Microsystems, Wetzlar, Germany), and the sections were then viewed on a Tecnai 20 TEM (Thermo Fisher Scientific, Waltham, MA, USA) at 120 kV. They were then double stained with UranyLess (EMS, Hatfield, PA, USA) for 2 min and with 3% lead citrate (EMS, Hatfield, PA, USA) for 1 min. Images were captured with a US1000X-P camera 200. The acquired images were stitched together using Photomontage software (Thermo Fisher Scientific, Waltham, MA, USA).

Ultrastructural analysis was conducted using transmission electron microscopy (TEM). Cells in each group were grown on coverslips. The samples were immediately fixed with 2.5% glutaraldehyde-mixed 2% paraformaldehyde solution for 1 h, followed by post-fixation in 2% osmium tetroxide (OsO₄; 19208, Electron Microscopy Sciences, Hatfield, PA, USA) for 1 h at 4 °C. The block was stained in 2% uranyl acetate (15200, Electron Microscopy Sciences, Hatfield, PA, USA) and dehydrated with a graded ethanol series. The samples were then embedded into epoxy medium (EMS, Hatfield, PA, USA). Embedded samples were sectioned (60 nm) with an ultra-microtome (UC7, Leica Microsystems, Wetzlar, Germany), and the sections were then viewed on a Tecnai 20 TEM (Thermo Fisher Scientific, Waltham, MA, USA) at 120 kV. They were then double stained with UranyLess (22409, Electron Microscopy Sciences, Hatfield, PA, USA) for 2 min and 3% lead citrate (22410, Electron Microscopy Sciences, Hatfield, PA, USA) for 1 min. Images were captured with a US1000X-P camera 200. The acquired images were stitched together using Photomontage software (Thermo Fisher Scientific, Waltham, MA, USA). The number of abnormal autophagic vacuoles of larger size was counted manually.

As described previously^{47 (link)}, extracellular parasites were pelleted in PBS. Conoid extrusion was induced by incubation with 40 µL of PBS/BIPPO for 5 min at 37 °C. 5 µL of the sample was applied on glow-discharged 200-mesh Cu electron microscopy grid for 5 min. The excess of the sample was removed by blotting with filter paper and immediately washed three times on drops of double distilled water. Finally, the sample was negatively stained with 0.5% aqueous solution of phosphotungstic acid (PTA) for 20 s then blotted with filter paper and air dried. Electron micrographs of parasites apical poles were collected with Tecnai 20 TEM (FEI, Netherland) operated at 80 kV acceleration voltage equipped with side mounted CCD camera (MegaView III, Olympus Imaging Systems) controlled by iTEM software (Olympus Imaging Systems).
For quantification, the preparation of the samples was performed in a blind manner. After acquisition, the names of TEM images were randomized using inhouse written MatLab script and these randomized sets of images were assessed by naive users to assign the presence or absence of the intraconoidal microtubules.

Purified AtTCP21 protein was dropped onto a carbon-coated copper grid that was glow-discharged into the air using a plasma cleaner (Harrick Plasma, Ithaca, NY, USA). After 3 min, the liquid solution was removed by touching the filter paper and the sample was negatively stained with 2% (w/v) uranyl acetate. The grids were examined using a 200 kV FEI Tecnai 20 TEM (FEI, Hillsboro, OR, USA) equipped with a Gatan CCD camera.

The coverslip was removed by thermal shock by liquid nitrogen dipping. The eggs of interest were located by comparison to the optical images acquired prior to embedding. The blocks were trimmed by a razor blade before diamond knife ultramicrotome (Leica UC7) sectioning 50–100 μm deep. One Pioloform-coated slot grid of four to eight 80 nm sections was collected followed by grids of 300 nm sections. A montage covering 25% of the egg was imaged (Tecnai 12 TEM, FEI) for the 80 nm sections for initial interpretation (data not shown). The 300 nm sections were coated with 25 nm gold fiducial markers (Aurion) followed by TEM tilt series tomography (Tecnai20 TEM, FEI; 200 keV) of spaces between yolk granules and any other features of interest. The single tilt series was taken ± 55° at 2° intervals at 2.1 nm/pixel. The series were aligned and back projection reconstructed (IMOD) prior to analysis in FIJI (35 (link)–37 (link)). A total of more than 20 tomograms were reconstructed with three to five representatives for each condition. The remaining resin blocks were prepared for SBF-SEM acquisition.