Jem 1230 transmission electron

Cell pellets (4000 g; 20 min) were processed as previously described [59 (link)]. The grids were washed three times in PBST, immunostained with anti-pGSN antibody (Supplementary Table 4), rinsed in distilled water, stained with uranyl acetate and lead citrate, and photographed with a Jeol JEM 1230 transmission electron microscope (Japan).

Purified samples were analysed by electron microscopy after being adsorbed to glow‐discharged carbon‐coated grids and stained with 2% (w/v) uranyl acetate as described previously (Núñez‐Ramírez et al., 2020 ). Grids were observed using a JEOL JEM‐1230 transmission electron microscope, operated at 100 kV, at a nominal magnification of 40,000. EM images were taken under low dose conditions with a CMOS Tvips TemCam‐F416 camera at 2.84 Å per pixel.
Image processing was performed using the SCIPION package (de la Rosa‐Trevín et al., 2016 (link)). The contrast transfer function of the microscope for each micrograph was estimated using CTFFIND4 (Rohou & Grigorieff, 2015 (link)). Particles were then automatically picked using the Xmipp routine inside SCIPION (Abrishami et al., 2013 (link)) and subjected to 2D classification using RELION (Scheres, 2012 (link)).

Lung fibroblasts were fixed in 2.5% glutaraldehyde with 0.1 M phosphate (pH 7.2) for 2 h, followed by the fixation with 1% osmium tetroxide for 1 h. 10 nm sections were sliced and stained with 2% uranyl acetate after dehydration in graded ethanol. JEM1230 transmission electron microscope (JEOL, Tokyo, Japan) was performed to determine the lipid drops in lung fibroblasts.

A drop of 4T1- or HC-11 conditioned medium was placed onto Formvar/carbon-coated grids and allowed to settle for 5 min. Each sample was then stained with a 2% uranyl acetate solution as a negative stain for extracellular vesicles. Following staining, the grids were dried and visualized under a JEM-1230 transmission electron microscope (JEOL USA, Peabody, MA, USA). Samples were visualized under vacuum with an electron beam with an accelerating voltage of 80 000 V. The resulting images are displayed at 80 000x magnification.

After designated treatment, HNE1/DDP cells were fixed with 2% glutaraldehyde in 0.1 M PBS (pH 7.3) for 2 h at 4°C and washed extensively with 0.1 M cacodylate buffer including 0.1% CaCl₂. Samples were fixed in 0.1 M cacodylate buffer containing 0.1% CaCl₂ for 30 min and then dehydrated through a graded series of ethanol and polymerized at 60°C for 48h. After being cut by ultracut microtome, the sections were stained with uranyl acetate and lead citrate. Subsequently, a JEM 1230 transmission electron microscope (JEOL, USA) was used to examine the section samples at a voltage of 60 kV.