Jem 1010

The heart tissues, about 1.0 mm × 1.0 mm × 1.0 mm in each group, were fixed with 2.5% glutaraldehyde overnight and washed three times with 0.1 mol/l (pH 7.2) phosphate buffer, then fixed with 1% osmium tetraoxide at 4 °C, washed with 0.1 mol/l phosphate buffer again, and dehydrated by ethanol at different concentrations. Epon812 resin/acetone (1:1) was applied to immerse samples for 1.5 h. Then, samples were soaked in fresh Epon812 resin for 30 min, and embedded for convergence overnight at 70 °C [18 (link)]. The tissue was cut into 60–80 nm thick slices. The ultrastructure of cardiac muscle cells was observed on transmission electron microscopy (JEM-1010, Hitachi, Tokyo, Japan).

Samples were processed as described previously (Théry et al., 2006 (link)) with several modifications. Briefly, 8 μl of EV‐enriched samples were fixed in 2% Paraformaldehyde (PFA) for 30 min, and deposited on Formvar‐carbon coated EM grids for 15 min. Then, samples were washed with PBS and post‐fixed with 1% Glutaraldehyde for 5 min, washed with distilled water, and then contrasted in a mixture of uranyl acetate (1%) and methyl cellulose (0,5%). Samples were analyzed either with a Jeol JEM1010 or a Hitachi HT7800 transmission electron microscope (Servicio Central de Soporte a la Investigación Experimental (SCSCIE), Universitat de València). JEM1010 was operated at 80 kV and images were recorded on a MegaView III digital camera. EV size was determined using the Olympus Image Analysis Software. Hitachi HT7800 was operated at 100 kV and images were recorded on a CMOS EMSIS XAROSA digital camera, and EV size was determined using the ImageJ software (Schneider et al., 2012 (link)).

Immunogold labelling of EVs was carried out at the Prince Felipe Research Centre of Valencia, Spain (CIPF) using EV‐enriched samples fixed in 2 % Paraformaldehyde in PBS for 30 min, and carbon‐coated nickel grids. Grids containing the EVs were washed in PBS, and blocked with PBS/0.1 M glycine/ 0.3 % BSA for 10 min. Grids were then incubated for 1 h with rabbit sera containing polyclonal antibodies (1:50) raised against F. hepatica TSG101 and the external domains of tetraspanin‐CD63 receptor (CD63rec) as primary antibodies (de la Torre‐Escudero et al., 2019 (link)), kindly provided by Dr. Mark Robinson, Queen's University, Belfast. After a washing with blocking solution for 20 min, the grids were incubated for 1 h with the gold‐labelled secondary antibodies (1:20) goat anti‐rabbit coupled to 12 nm gold particles (Abcam). Samples incubated with secondary antibodies alone were used as a control. After washing with PBS/0.1 M glycine, the grids were negative stained as described above and then viewed using a Jeol JEM1010 or a Hitachi HT7800 transmission electron microscope as previously described.