Tem em 910

The specimens were divided into two fragments, one for histological analyses by light microscopy and the other for morphological analyses by transmission electron microscopy (TEM). For TEM analyses, the lymphatic vessels and veins were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4), containing 0.05% (w/v) Alcian Blue 8GX, at 4 °C over-night and post-fixed in 2% buffered osmium tetroxide for 1 h. The specimens were then dehydrated with graded concentrations of acetone and embedded in Araldite epoxy resin (Durcupan ACM, Fluka, Sigma-Aldrich Co., St. Louis, MO, USA) according to standard protocols.
For orientation, semi-thin sections (1.5 μm) were cut on a Reichert Ultracut S ultramicrotome using glass knives and stained with a 1% aqueous solution of toluidine blue and examined with an optical microscope (Nikon Eclipse E800). Ultrathin sections (90 nm) were prepared with an ultramicrotome (Reichert UltracutS) and counterstained with uranyl acetate in saturated solution and lead citrate according to Reynolds and observed under transmission electron microscope (TEM Zeiss EM 910, Zeiss, Wetzlar, Germany) at 80,000× magnification. The depth of the glycocalyx was measured near the surface, where the plasma membrane was visible.

Both GD and GDBio ADNVs fractions were prepared for Transmission Electron Microscopy (TEM), imaging following a procedure for the staining of extracellular vesicles described by Corona et al., 2023 [31 ]. Briefly, EVs were deposited at a concentration of 10⁹ particles/mL and a TEM Grids 200 Mesh Cu/Pd. Sample and grid were fixed with 2% paraformaldehyde (PFA) 1% glutaraldehyde in 100 mM PBS, pH 7,4 solution. For staining, a contrasting solution of Methylcellulose/Uranyl Acetate was applied for 10 min. The grid was dried, and pictures of the grid were taken with TEM Zeiss EM 910 instrument (Carl Zeiss Microscopy, Oberkochen, German). Images were recorded by a CCD digital camera (Ultrascan 1000, Gatan, Munich, Germany).

Exosome fixation for TEM was performed using 2% glutaraldehyde solution in phosphate buffer (ratio 1:1). After the fixation phase, exosomes were deposited, rinsed, and stained with heavy metal compounds onto a gridded slide following standard protocols. The visualization of the slide was performed using a TEM Zeiss EM 910 instrument (Zeiss, Oberkochen, Germany).