Tem center

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.

The cells were processed for transmission electron microscopy as described previously [23 (link)]. Briefly, the cells were fixed in 3% glutaraldehyde (in 0.1 M cacodylate buffer, pH 7.2; Sigma) for 3 h at room temperature. Subsequently, the cells were washed in cacodylate buffer (0.1 M, pH 7.2) and post-fixed in 1% OsO4 (in 0.1 M cacodylate buffer, pH 7.2; Sigma) for 1 h at room temperature. After the rinsing procedure, the cells were dehydrated in graded alcohols (50%, 75%, 96%, and 100%), clarified in propylene oxide, and embedded in a mixture of Epon 812 and Durcupan (Sigma; polymerization for 3 days at 60 °C). Toluidine blue was used to stain the semithin sections. Ultrathin sections were cut on Ultrotome Nova (LKB, Vienna, Austria). These sections were then collected onto formvar carbon-coated copper grids, counterstained with uranyl acetate and lead citrate, and examined under a JEOL JEM-1400Plus transmission electron microscope (at 120 kV, JEOL, Tokyo, Japan). The images were taken with the integrated 8Mpix CCD camera and processed further using the software TEM Center (Ver. 1.7.3.1537, JEOL, Tokyo, Japan).