Aqueous uranyl acetate

Lc3b-AP2-expressing imMEFs were grown on aclar sheets (Science Services), supplemented with 4.83 µM Hemin chloride (ROTH) for 16 h and treated with 200 nM BafA1 (Biomol) for 2 h before fixation. Cells were fixed in 2.5% glutaraldehyde (EM-grade, Science Services) in 0.1 M sodium cacodylate buffer (pH 7.4; CB) for 30 min. Fixation and the following processing steps were carried out on ice. After washes in CB, endogenous peroxidases were blocked in 20 mM glycine (Sigma) in CB for 5 min and cells washed in CB. 1x diaminobenzidine (DAB) in CB with 2 mM calcium chloride was prepared from a 10x DAB stock (Sigma) in hydrochloric acid (Sigma) and added to the cells for 5 min without and for another 40 min with 10 mM H₂O₂ (Sigma). After washes in CB, cells were postfixed in reduced osmium (1.15% osmium tetroxide, Science Services; 1.5% potassium ferricyanide, Sigma) for 30 min, washed in CB and water and incubated over-night in 0.5% aqueous uranylacetate (ScienceServices). Dehydration was accomplished using a graded series of ice-cold ethanol. Cell monolayers were infiltrated in epon (Serva) and cured for 48 h at 60 °C. Cells were ultrathin sectioned at 50 nm on formvar-coated copper grids (Plano). TEM images were acquired on a JEM 1400plus (JEOL) using the TEMCenter and Shotmeister software packages (JEOL) and analysed in Fiji.

Paraformaldehyde-fixed root pieces (from June 2014) that were previously used for FISH (see above) were also used for scanning transmission electron microscopy (STEM) imaging. Thin sections (~70 nm) were prepared with the Leica UC7 Ultramicrotome (Ultracut UC7, Leica Microsystems) using a diamond knife, mounted on formvar-coated slot-grids (Agar Scientific). Sections were stained with osmium tetroxide (OsO₄), followed by 0.5% aqueous uranyl acetate (Science Services) for 20 min and 2% Reynold’s lead citrate for 6 min, with three washing steps between each step. Sections were imaged at 20–30 kV using the Quanta 250 FEG scanning electron microscope (FEI) equipped with a STEM detector using the xT microscope control software (v.6.2.6).

Eyes were enucleated and fixed with Karnovsky’s fixative (1% paraformaldehyde, 3% sodium cacodylate–HCl, and 3% glutaraldehyde; Sigma-Aldrich, Buchs, Switzerland) for 24 h. After washing three times in TEM buffer (2.5% glutaraldehyde and 0.1 M sodium cacodylate–HCl), the eyes were post-fixed in 4% osmium tetroxide in cacodylate buffer for 15 min. Samples were dehydrated through a graded series of acetone, washed with a resin/1,2-propylene oxide mixture (Merck, Darmstadt, Germany), and embedded in epoxy-based resin. Ultrathin sections (80 nm) were cut with an ultramicrotome Ultracut E (Reichert Microscope Services, Depew, NY, USA) equipped with a 45° diamond knife (Diatome, Biel, Switzerland). Sections were placed onto copper grids (G100H-C3; Science Services, Munich, Germany) and counterstained with 4% aqueous uranyl acetate and 0.1% Reynolds’ lead citrate (Science Services). TEM analyses were performed on a CM 12 electron microscope (Philips Applied Technologies, Eindhoven, Netherlands).