Agar low viscosity resin lvr

Freshly moulted and inter-moult specimens of C. acosta were treated as described above, including the complete dehydration step in acetone before being embedded into Agar Low Viscosity Resin (LVR, Agar Scientific, Stanstead, Essex, UK). Cured resin blocks were sectioned at 1 μm (semithin) or 60 nm (ultrathin) on a Leica UC6 ultramicrotome (Leica Microsystems, Wetzlar, Germany). Semithin sections were stained with toluidine blue for few seconds on a heating plate at 60 °C, whereas ultrathin sections were contrasted with uranylacetate for 30 min and leadcitrate for 5–10 min. Light microscopy sections were viewed with an Olympus BX53 equipped with a DP73 microscope camera (Olympus, Tokyo, Japan). Ultrathin sections were analyzed with a Zeiss Libra120 TEM (Zeiss, Oberkochen, Germany).

Pellets comprising EVs obtained by ultracentrifugation were prepared for TEM following collection either directly or from the resulting pellet of binding to 4 μm CD63‐coated latex beads. EVs were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) for 1 hr. Following post‐fixation with 1% osmium tetroxide (0.1 M sodium cacodylate buffer, pH 7.4, at room temperature for 1 hr), the pellet was washed for 3 × 10 min. in 0.1 M sodium cacodylate buffer (pH 7.4). The sample was dehydrated by several washes of increasing ethanol concentration. Finally, the sample was washed twice with dry 100% ethanol for 30 min. A 1:1 ratio of dry ethanol to Agar low viscosity resin (LVR) (Agar Scientific, Essex, UK) was added. After 30 min., further Agar LVR was added to a final 1:2 ratio and left overnight under gentle movement to allow ethanol to evaporate off. The Agar LVR resin was polymerized at 60°C until the resin hardened. Ultrathin sections were obtained on a Leica Ultracut and collected on copper grids (Agar Scientific). Grids were stained with saturated alcoholic uranyl acetate and aqueous lead citrate. Stained sections were viewed with a JEOL^® 100CXII transmission electron microscope.