Tecnai 12 120 kv microscope

Samples for EM analyses were prepared using standard methods, as described previously (Rizzo et al., 2013 (link)). In brief, the cells were fixed with 4% paraformaldehyde and 0.05% glutaraldehyde for 30 min at room temperature for immunonanogold labeling or 1% glutaraldehyde for 2 h for morphological analyses. For immunonanogold labeling the cells were permeabilized with 0.1% saponin in blocking solution (PBS containing 1% BSA and 50 mM NH₄Cl) and incubated with antibodies to antigens of interest in the same solution at 4°C overnight (anti-IMPDH2 1:1000 and anti-ARL2 1:10), followed by secondary antibodies labeled with nanogold in the same solution for 2 h at room temperature. The gold particles were then enhanced by nanogold enhancer (Nanoprobes) according to the manufacturer’s instructions. The samples were then embedded in epon and ultrathin sections (50–70 nm) were prepared. The samples were examined using a Tecnai 12 120 kV microscope (FEI, The Netherlands). For quantification of the association of RRs with cellular organelles, the criteria used were the following: the membrane of the organelle should be within 20 nm of a RR and the length of this close association should be continuous for at least 50 nm.