Gold enhancement solution

Cells were fixed in 4% paraformaldehyde, washed, permeabilized for 10 minutes with 0.25% saponin, 0.1% BSA in PBS, and blocked (0.2% BSA, 5% Normal Goat Serum, 50mM NH₄Cl, 0.1% saponin, 20mM phosphate buffer, 150mM NaCl). Coverlips were then incubated with primary antibody (anti-UBC9 BD Biosciences or anti-LC3 Novus Biologicals) for 2 hours, washed (0.1% BSA, 0.1% Saponin in PBS) and incubated for 1 hour with secondary antibodies conjugated with nanogold (Nanoprobes). Samples were then fixed with 1% glutaraldehyde for 1 hour and nanogold was enlarged with a gold enhancement solution (Nanoprobes) according to manufacture instructions, postfixed (1% OsO₄, 1.5% potassium ferrocyanide in 0.1M cacodylate buffer pH 7.4), enbloc stained with 1% uranyl acetate over/night at 4°C, dehydrated with ethanol, embedded in EPON 812 and cured in an oven at 60°C for 48 hours. Ultrathin sections (70-90nm) were cut on an ultramicrotome (Leica FC7, Leica microsystem). Grids were stained with uranyl acetate and Sato’s lead solutions and observed in a Leo 912AB Zeiss Transmission Electron Microscope (Carl Zeiss). Digital micrographs were taken with a 2Kx2K bottom mounted slow-scan Proscan camera (ProScan, Lagerlechfeld, Germany) controlled by the EsivisionPro 3.2 software (Soft Imaging System).

Cells were fixed in 4% PFA EM grade and 0.2 M HEPES buffer for 1 h at RT or in Periodate-lysine-paraformaldehyde (PLP) for 2 h at RT. After three washes in PBS, cells were incubated 10 min with 50 mM glycine and blocked 1 h in blocking buffer (0.2% bovine serum albumin, 5% goat serum, 50 mM NH₄Cl, 0.1% saponin, 20 mM PO₄ buffer, 150 mM NaCl). Staining with primary antibodies and nanogold-labeled secondary antibodies (Nanoprobes) were performed in a blocking buffer at RT. Cells were fixed for 30 min in 1% GA and nanogold was enlarged with gold enhancement solution (Nanoprobes) according to the manufacturer’s instructions. Cells were post-fixed with OsO₄ and processed as described for conventional EM. Images were acquired with Talos L120C TEM (FEI, Thermo Fisher Scientific) operating at 120 kV. Images were acquired with a Ceta CCD camera (FEI, Thermo Fisher Scientific) using Velox 3.6.0 (FEI, Thermo Fisher Scientific).

Wildtype and AMACO-deficient newborn mouse back skin was excised and carefully sliced into 1 mm cubes and stored in either Dulbecco’s Modified Eagle Medium (DMEM) or Michel’s Buffer. The skin samples were subsequently incubated in a solution of a polyclonal affinity-purified antibody (Table 1), diluted 1:5 in serum-free DMEM, overnight at 4 °C. The tissue cubes were washed in DMEM for 4 h and subsequently immersed in a 1 nm gold-labeled diluted secondary antibody suspension (Aurion GAR ultrasmall, Electron Microscopy Sciences, Hatfield, Pa), overnight at 4 °C. After a rigorous wash in DMEM, the tissues were immersed in gold enhancement solution (Nanoprobes, Yaphank, NY), rinsed, fixed in 1.5% glutaraldehyde, 1.5% paraformaldehyde with 0.05% tannic acid, post-fixed in 1% osmium tetroxide, and then dehydrated and embedded in Spurr’s epoxy. The stained sections were examined using either a Philips EM410LS or a FEI Tecnai G2 transmission electron microscope (Thermofisher, Hillsboro, Or).