Tecnai t12 electron microscope

The ligase complex reconstituted in nanodiscs or in digitonin at a concentration of 0.02 mg ml^–1 was applied to glow-discharged continuous carbon grids (Electron Microscopy Sciences, Inc.). After 1 min of adsorption, the grids were blotted with filter paper to remove excess sample, immediately washed twice with 4 μl of 1.5% freshly made uranyl formate solution and incubated with 4 μl of 1.5% uranyl formate solution for an additional 30 s. The grids were then further blotted with filter paper to remove the uranyl formate solution, air dried at room temperature and examined with a Tecnai T12 electron microscope (Thermo Fisher Scientific) equipped with an LaB6 filament and operated at 120 kV acceleration voltage, using a nominal magnification of ×52,000 at a pixel size of 2.13 Å.

Cells were grown on a μ-Slide 8 Well Grid-500 (80826-G500, ibidi). For light microscope (LM) imaging, cells were observed under FEI Corrsight microscope equipped with Andromeda spinning disk. After LM imaging, cells were fixed with 2.5% Glutaraldehyde in 0.1 M phosphate buffer for 24 h at 4 °C. Then cells were washed with 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide in the same buffer containing 1.5% potassium ferricyanide for 1 h in the dark at 4 °C. Samples were dehydrated in ethanol, infiltrated with Epon resin for 2 days, embedded in the same resin and polymerized at 60 °C for 48 h. Ultrathin sections were cut using a Leica Ultracut UCT ultramicrotome (Leica Microsystems Vienna) and mounted on Formvar-coated copper grids, then stained with 2% uranyl acetate in water and lead citrate. Sections were observed in a Tecnai T12 Electron Microscope equipped with an Eagle 4kx4k CCD camera (Thermo Fisher Scientific, The Netherlands). Quantification of mitochondria related parameters from transmission electron microscopy (TEM) cross-sections, including mitochondrial length, perimeter, area, and cristae number per mitochondrion were done as previously reported [82 ] with Fiji.

After hypoxia exposure (24 h), cells are exposed to BSA‐gold (10 nm) (dept. of cell biology, University Medical Centre, Utrecht) for 10 min. After BSA‐gold internalisation (10 min), cells were fixed with 2.5% glutaraldehyde in phosphate buffer. Cells were kept in the fixative during 24 h at 4°C. Then, they were washed with 0.1 M cacodylate buffer and potsfixed with 1% osmium tetroxide in the same buffer containing 1.5% potassium ferricyanide during 1.5 h at 4°C. Then the samples were embedded in agarose and dehydrated in ethanol, infiltrated with Epon resin during 2 days, embedded in the same resin and polymerised at 60°C during 48 h. Ultrathin sections were obtained using a Leica Ultracut UCT ultramicrotome and mounting on Formvar‐coated copper grids. They were stained with 2% uranyl acetate in water and lead citrate. Then, sections were observed in a Tecnai T12 electron microscope equipped with an Eagle 4kx4k CCD camera (Thermo Fisher Scientific, The Netherlands).

Organoids were removed from BME, washed with excess AdDF+++ + 5% FBS. Organoids were fixed with 1.5% glutaraldehyde in 0.1 M cacodylate buffer. They were kept in the fixative for 24 h at 4°C. Then, they were washed with 0.1 M cacodylate buffer and postfixed with 1% osmium tetroxide in the same buffer containing 1.5% potassium ferricyanide for 1 h (dark) at 4°C. Then, the samples were dehydrated in ethanol, infiltrated with Epon resin for 2 days, embedded in the same resin and polymerized at 60°C for 48 h. Ultrathin sections of 50 nm were obtained using a Leica Ultracut UCT ultramicrotome (Leica Microsystems, Vienna) and mounted on Formvar‐coated copper grids. They were stained with 2% uranyl acetate in water and lead citrate. Then, sections were observed in a Tecnai T12 electron microscope equipped with an Eagle 4kx4k CCD camera (Thermo Fisher Scientific, the Netherlands).