Em grade

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 microglial pellet was preserved throughout all fixation, contrasting and infiltration steps. Cells were fixed for 30 min in 2% PFA/2.5% glutaraldehyde (EM-grade, Science Services) in 0.1 M sodium cacodylate buffer (pH 7.4) (Sigma Aldrich), washed three times in 0.1 M sodium cacodylate buffer before postfixation in reduced osmium (1% osmium tetroxide (Science Services)/0.8% potassium ferrocyanide (Sigma Aldrich) in 0.1 M sodium cacodylate buffer). After contrasting in aqueous 0.5% uranylacetate (Science Services), the pellet was dehydrated in an ascending ethanol series, infiltrated in epon (Serva) and cured for 48 h at 60 °C. Ultrathin sections (50 nm) were deposited onto formvar-coated copper grids (Plano) and postcontrasted using 1% uranyl acetate in water and ultrostain (Leica). Transmission Electron Microscopy micrographs were acquired on a JEM 1400plus (JEOL) using the TEMCenter and tile scans with the ShotMeister software packages (JEOL), respectively. Three independent cell preparations were analyzed (n = 3).

293T cells expressing APEX2-tagged TECPR2 WT and L440Rfs were grown on aclar sheets (Science Services) and fixed with 2.5% glutaraldehyde (EM-grade, Science Services) in 0.1 M pH 7.4 sodium cacodylate buffer (CB) for 30 min on ice. Endogenous peroxidases were blocked with 20 mM glycine (Sigma) for 5 min on ice and cells washed in CB. Cells were saturated with freshly prepared 1× diaminobenzidine (DAB, in CB supplemented with 2 mM calcium chloride) for 5 min and APEX2 activity was triggered with 1× DAB supplemented with 10 mM H₂O₂ (Sigma) for 40 min on ice. Cells were washed with CB and subsequently postfixed and contrasted in reduced osmium (1.15% osmium tetroxide (Science Services) 1.5% potassium ferricyanide (Sigma)) for 30 min. After washes in CB and H₂O₂, cells were incubated in 0.5% aqueous uranylacetate (Science Services) over-night and dehydrated using a graded series of ice-cold ethanol-water composite. Cell monolayers were infiltrated in epon (Serva) and cured for 48 h at 60 °C. 50 nm ultrathin sections were generated on formvar-coated copper grids (Plano). Sections were imaged using a JEM-1400 + (JEOL) equipped with a XF416 (TVIPS) and the EM-Menu software (TVIPS) and analyzed using ShotMeister (JEOL).