Bsa c

Ten microliters of EVs were adsorbed to carbon‐coated formvar grids for 20 min at room temperature. Grids were washed with PBS and blocked with blocking buffer (0.5% fish skin gelatin (Sigma) and 0.1% BSA‐c (Aurion) in PBS) for immunogold labelling or fixed using 1% glutaraldehyde in PBS. Immunogold labelling was performed by staining with rabbit‐anti‐mouse IgG (Rockland, cat. 610–4120, 1:250) followed by 10 nm Protein A‐gold (CMC, Utrecht, The Netherlands) and fixation using 1% glutaraldehyde in PBS. After excessive washing with MilliQ water, grids were counterstained with uranyl‐oxalate and embedded in methyl cellulose uranyl‐acetate. Imaging was performed using a Tecnai T12 transmission electron microscope (FEI). For gold quantification, 5–10 pictures were acquired at random grid locations at a magnification of 49,000×. A custom‐made ImageJ script based on intensity thresholding was run on each picture to count the total number of gold particles. Membrane‐associated gold was counted manually using the multi‐point tool in ImageJ and expressed as a percentage of total gold.

Immuno-EM experiments were performed by Colzyx AB (www.colzyx.com). In brief, WT or RagA/B KO MEF cells were treated as described in the figure legends and collected by centrifugation (5 min, 250g). Each sample (5 × 10⁶ cells) was gently transferred into a 1 ml sample tube and centrifuged at 2,500g for 20 min. Pellets were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.2) for 2 h at 4 °C and subsequently washed with 0.15 M cacodylate (pH 7.2). Samples were then post-fixed with 1% osmium tetroxide and 0.15 M sodium cacodylate (pH 7.2) for 1 h at 4 °C, washed 3× with 1 ml 0.15 M sodium cacodylate (pH 7.2), and further processed for transmission electron microscopy according to standard protocols. Briefly, the fixed and washed samples were dehydrated in ethanol and further processed for routine Epon embedding. Sections were cut with an LKB ultratome equipped with a diamond knife and mounted on Formvar-coated nickel grids.
Before immunostaining, grids were subjected to antigen unmasking with sodium metaperiodate as described previously^{107 (link)}. Grids were incubated in a humidified chamber on 100 μl drops of a saturated sodium metaperiodate aqueous solution for 1 h at room temperature. For immunostaining, the grids were floated on 100 μl drops of immune reagents displayed on a sheet of Parafilm in a humidified chamber. Free aldehyde groups were blocked with 50 mM glycine, and the grids were then incubated with 5% donkey serum (ab7475, Abcam) in incubation buffer (0.2% BSA-c (#900.022, Aurion) in PBS, pH 7.6) for 15 min. The blocking procedure was followed by overnight incubation with the following primary antibodies at 4 °C: rabbit monoclonal anti-mTOR antibody against mTOR (#2983, CST; dilution 1:80) or rat monoclonal antibody against mouse LAMP2 (#ABL-93, Developmental Studies Hybridoma Bank; dilution 1:80). After washing the grids in a large volume (200 ml) of incubation buffer, staining with the gold particle-conjugated antibodies (10 nm size for mTOR and 5 nm size for LAMP2) was performed by floating the grids on drops containing the gold conjugate reagents (diluted 1:20 in incubation buffer) for 60 min at room temperature. After additional washes (3× in 50 ml incubation buffer each), the sections were post-fixed in 2% glutaraldehyde. Finally, sections were washed with distilled water and post-stained with uranyl acetate and lead citrate. Specimens were observed in a Philips/FEI CM100 transmission electron microscope (Philips/FEI) operated at 80 kV accelerating voltage and images were recorded with a side-mounted Olympus Veleta camera (Olympus) with a resolution of 2,048 × 2,048 pixels.
For quantifications, values are shown as number of 10 nm gold particles per square micrometer. Localization of gold particles to lysosomes or the cytoplasm was assessed based on morphological criteria and LAMP2 staining. Twenty randomly selected areas were evaluated per sample from three independently processed ultrathin sections (a total of 58–60 cellular profiles) per experimental condition.