Uc7 ultramicrotome

To assess the ultrastructure of the obtained EVs, we fixed it in 2.5% glutaraldehyde. After 12 h from the start of fixation, the samples were placed in a 1% OsO₄ solution in phosphate buffer with the addition of sucrose, dehydrated and embedded in Epon-812 (Fluka, Charlotte, NC, USA). Ultrathin sections of 0.1 µm thickness were prepared on a Leica UC7 ultramicrotome (Leica, Wetzlar, Germany) and mounted on copper grids (Sigma). Sections were counterstained with uranyl acetate and lead citrate and then examined using a Hitachi 7700 transmission electron microscope (Hitachi, Tokyo, Japan).

The STEM experiments were adapted from previously described experiments (Kuipers et al, 2015 (link)). Briefly, paraffin embedded cortical sections of AM and AM;Serf2^br−/− were deparaffinized and postfixed with 1% osmium tetroxide/1.5% potassium ferrocyanide in 0.1 M sodium cacodylate, dehydrated through ethanol, and embedded in EPON (Serva) using a tissue processor (EM TP 709202; Leica). Ultrathin sections (80 nm) were cut using the Leica uc7 ultramicrotome and collected on formvar-coated cupper grids (electron microscopy sciences). A large area scan using scanning transmission detection was made using a Zeiss supra55 SEM with ATLAS. STEM detection with a four-quadrant STEM detector was used in inverted dark-field mode, at 28 kV with 30 μm aperture at 3.5 mm working distance. All images were recorded at the same scan speed (cycle time 1.5 min at 3,072 × 2,304 pixels). Contrast and brightness were set based on a live histogram. High-resolution large-scale STEM images at 2.5 nm pixel size were generated with the external scan generator ATLAS (Fibics), individual tiles were stitched in VE viewer (Fibics), exported as a html file, and uploaded to the website www.nanotomy.org.

Muscle samples were washed in 0.15 M cacodylate buffer and postfixed for 2 h in 2% osmium tetraoxide in 0.1 M cacodylate buffer. After washing in cacodylate buffer and water, samples were dehydrated in increasing concentrations of ethanol, with the final dehydration occurring in 100% acetone and using propylene oxide. To avoid humidity, the samples were infiltrated with 1/3 Epon and 2/3 propylene oxide. Finally, they were infiltrated 3 times with pure Epon and then placed into molds with fresh Epon at 60 °C for 24 h. Semi-thin (0.5 µm thick) and ultrathin (70 nm thick) transverse sections were cut with a Leica UC7 ultramicrotome (Leica, Leica Microsystemes SAS, Nanterre, France). The semi-thin sections were stained with 1% toluidine blue in 1% borax, and the region of interest was selected under the microscope. The ultrathin section of the selected region was collected using copper grids (200 mesh, EMS, Souffelweyersheim, France) and contrasted with Reynold’s lead citrate. The ultrathin sections were observed using a Hitachi HT7700 electron microscope (Milexia, Saint Aubin, France) operating at 100 kV. Pictures (2048 × 2048 pixels) were taken with an AMT41B camera.

Mice were anesthetized using isoflurane before decapitation and optic nerve extraction. Optic nerves were immediately transferred to a fixative solution (4% PFA, 2.5% glutaraldehyde in phosphate buffer with 0.5% NaCl, pH 7.4) and fixed overnight at 4 °C. Tissue preparation and EM were carried out as previously described^{111 (link)}. Briefly, following postfixation with 2% OsO₄ (Science Services) in 0.1 M phosphate buffer (pH 7.3) and acetone dehydration, tissue fragments were embedded in EPON (Serva). Ultrathin sections were cut with a Leica UC7 ultramicrotome (Leica) and then stained using UranyLess (Science Services). EM pictures were captured with a Zeiss EM912 electron microscope (Zeiss) equipped with an on-axis 2k charge-coupled device camera (TRS). EM image analysis was performed using ImageJ (Fiji, version 1.52p). Axonal diameters and g ratios (ratio of axon diameter to the diameter including the myelin sheath) were analyzed using five random overview EM pictures (at 8,000× magnification), with 250 axons evaluated per animal. Calculations were based on circular areas equivalent to the measured areas. For axonal pathology assessment, eight to ten EM images per animal were analyzed. The experimenters were blinded to the genotypes.

Seedlings were fixed in cold 90% acetone for at least 30 min at 4 °C. Acetone was removed, and the material was washed twice with rinse solution (0.5 M Na₂HPO₄, 0.5 M NaH₂PO₄, 0.1 M K₃Fe(CN)₆, 0.1 M K₄Fe(CN)₆). The rinse solution was removed, and stain solution added (rinse solution complemented with 2 mM x-Gluc (5‐bromo‐4‐chloro‐3‐indolyl‐beta‐D‐glucuronide). Samples were then gently vacuum-infiltrated for 30 min and then incubated at 37 °C, in the dark, for 30 min or 24 h. Samples were then washed in water and cleared in chloral hydrate (8 g choral hydrate, 3 mL 100% glycerol, 1 mL dH₂O) for at least 45 min, before mounting in chloral hydrate.
Embedding primary roots GUS stained for sectioning was performed using Technovit 7100 according to the manufacturer’s instruction (Electron Microscopy Science). Sectioning was performed using a Leica UC7 (Ultra-microtome). Images were obtained with a DM6000 microscope (Leica).