Megaview 2

BMDMs were scraped and fixed in 2 % glutaraldehyde for 2 h at 4 °C, postfixed in 1% Osmium tetroxide for 1 h at 4 °C, dehydrated, and embedded in Epon. Samples were then cut using an RMC/MTX ultramicrotome (Elexience), and ultrathin sections (60–80 nm) were mounted on copper grids, contrasted with 8% uranyl acetate and lead citrate, and observed with a Jeol 1200 EX transmission electron microscope (Jeol LTD) equipped with a MegaView II high-resolution transmission electron microscopy camera. Pictures of cell sections were taken at ×45,000 magnification. Mitochondria number per section was measured to evaluate mitochondria density. For cristae analysis, mitochondria and cristae were outlined using ImageJ (Fiji) and both the total length and number of cristae in each mitochondrion were calculated, as previously described^{42 (link)}. For the analysis of mitochondria dynamics, the long and short axis of each mitochondrion, as well as their perimeter and area, were measured. From these values, aspect ratio (major axis/minor axis) and form factor (perimeter)²/(4×pixArea) were calculated. TEM analyses were performed in triplicate, and a minimum of 11 images per sample were taken.

Transmission electron microscopy (TEM) was used to visualize EVs isolated from both yeast and hyphal-cell morphologies. Samples were fixed for 2 h at room temperature in a buffer containing 2.5% glutaraldehyde and 0.1 M cacodylate and then incubated overnight at 4°C in 4% paraformaldehyde, 1% glutaraldehyde, and 0.1% PBS. After that, the samples were treated with 2% osmium tetroxide (TAAB Laboratories, UK) for 90 min, serially dehydrated in ethanol, and embedded in EMBed-812 resin (Electron Microscopy Sciences). Thin sections (50 to 70 nm) were obtained by ultracut and observed in a JEOL JEM 1010 transmission electron microscope operating at 100 kV. Pictures were taken with a Megaview II camera. TEM images were analyzed with Soft Imaging Viewer software. TEM was carried out in the Electron Microscopy Facility (ICTS) of the Complutense University of Madrid (UCM).

Transmission electron microscopy (TEM) was used to visualize EVs isolated from both yeast and hyphal-cell morphologies. Samples were fixed for 2 h at room temperature in a buffer containing 2.5% glutaraldehyde and 0.1 M cacodylate and then incubated overnight at 4°C in 4% paraformaldehyde, 1% glutaraldehyde, and 0.1% PBS. After that, the samples were treated with 2% osmium tetroxide (TAAB Laboratories, UK) for 90 min, serially dehydrated in ethanol, and embedded in EMBed-812 resin (Electron Microscopy Sciences). Thin sections (50 to 70 nm) were obtained by ultracut and observed in a JEOL JEM 1010 transmission electron microscope operating at 100 kV. Pictures were taken with a Megaview II camera. TEM images were analyzed with Soft Imaging Viewer software. TEM was carried out in the Electron Microscopy Facility (ICTS) of the Complutense University of Madrid (UCM).