0.1 m sodium cacodylate buffer

Hepatopancreas tissue was fixed in 2.5% glutaraldehyde (Agar Scientific, Stansted, UK) in 0.1 M sodium cacodylate buffer (pH 7.4) (Agar Scientific, Stansted, UK) for a minimum of 2 h at room temperature and rinsed in 0.1 M sodium cacodylate buffer (pH 7.4). Tissues were post-fixed for 1 h in 1% osmium tetroxide (Agar Scientific, Stansted, UK) in 0.1 M sodium cacodylate buffer. Samples were washed in three changes of 0.1 M sodium cacodylate buffer before dehydration through a graded acetone series. Samples were embedded in Agar 100 epoxy (Agar Scientific, Agar 100 premix kit medium) and polymerised overnight at 60 °C in an oven. Semi-thin (1–2 µm) sections were stained with Toluidine Blue for viewing with a light microscope to identify suitable target areas. Ultrathin sections (70–90 nm) of these areas were mounted on uncoated copper grids (Agar Scientific, Stansted, UK) and stained with 2% aqueous uranyl acetate (Agar Scientific, Stansted, UK) and Reynolds’ lead citrate [25 (link)]. Grids were examined using a JEOL JEM 1400 transmission electron microscope and digital images captured using an Advanced Microscopy Techniques (AMT) XR80 camera and AMT V602 software.

Tissue was processed for electron microscopy as previously described (Czopka and Lyons, 2011 (link)). Briefly, 4 dpf larvae were anaesthetised with tricaine and chemically fixed with microwave stimulation using a solution of 2% glutaraldehyde, 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (Agar Scientific, Essex, UK). Embryos subsequently underwent secondary fixation with microwave stimulation in 2% osmium tetroxide in 0.1 M sodium cacodylate/imidazole (Agar Scientific) followed by en bloc stain in saturated uranyl acetate (~8%, w/v, in water) (TAAB Laboratories Equipment Ltd., Berkshire, UK). Samples were then progressively dehydrated in a series of ethanol and acetone washes before being embedded in Embed-812 resin (Electron Microscopy Sciences, Hatfield, PA). Ultra-thin sections (70 nm thickness) were cut around somite 15 using a Reichert Jung Ultracut microtome (Leica Microsystems, Wetzlar, Germany) and stained using uranyl acetate (TAAB Laboratories Equipment Ltd.) and Sato lead stain (see (Czopka and Lyons, 2011 (link)) for detailed protocol). Stained sections were imaged using a Jeol JEM-1400 Plus transmission electron microscope (JEOL USA, Inc., Peabody, MA) at 8600x magnification. Individual tiles were automatically aligned using the PhotoMerge tool in Adobe Photoshop CC (Adobe Systems Inc., San Jose, CA) and quantified manually using Fiji (ImageJ).

Fixed placental fragments were washed twice for 10 min in 0.1 m sodium cacodylate buffer (Agar Scientific, UK) at pH 7.4 containing 0.23 m sucrose (BDH, UK) and 2 mm CaCl₂ (BDH, UK). The specimens were then placed for 60 min in 2% osmium tetroxide (BDH, UK) in 0.1 m sodium cacodylate (Agar Scientific, UK) at pH 7.4, then washed three times for 10 min with distilled water. Samples were then treated with 2% aqueous uranyl acetate (Agar Scientific, UK) for 20 min. Samples were dehydrated using a graded ethanol series. Specimens were then treated with 50 : 50 Spurr resin : acetonitrile (Fisher, UK) overnight and infiltrated with fresh Spurr resin for 6 h. Finally, specimens were embedded in Spurr resin for 16 h at 60 °C.
Gold/silver ultrathin sections were cut using a Reichert Ultracut E ultramicrotome, stained with Reynolds lead citrate, and viewed by TEM to study structures associated with stromal vesicles and fibroblast‐like stellate cells (Tecnai 12, Thermo Fisher, Eindhoven).

5 × 10⁵ cells submitted or not to shear stress assay were centrifuged at 200g for 5 min in a 1.5 mL polypropylene tube. The pellet was incubated with a solution of Karnovsky fixative at 4 °C overnight. The pellet was washed with 0.1 M sodium cacodylate buffer (Agar Scientific, Stansted, UK), then post-fixed for 1 h in 1% osmium tetroxide (Agar Scientific) in sodium cacodylate buffer and then washed three times and finally re-suspended in 20 µL of cacodylate buffer. A 5 µL drop was introduced into the core of a warm (40–50 °C) 8% agarose fluid gel in a 1.5 mL polypropylene tube. Once solidified, a 3 mm³ block was cut around the drop. Dehydration process was performed by 3 successive baths of 10 min in 100% ethanol followed by incubation in propylene oxide. The sample was then embedded in Epon-Araldite and ultra-thin sections (65 nm with Leica EM-UC6 ultra-microtome) were stained for 10 min in 5% uranyl acetate and 5 min in lead citrate. The sections were imaged with a FEI CM120 transmission electron microscope at 120 kV, using a Gatan USC1000-SSCCD camera.

Transmission electron microscopy (TEM) of T/L 3D TE constructs (n = 3) was performed following fixation in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (Agar Scientific, Essex, UK) for 8 h, followed by a second fixation and contrast stain with 0.1% osmium tetroxide for 90 min. Samples were stained with 8% uranyl acetate in 0.69% maleic acid for 90 min, dehydrated in ascending ethanol concentrations, and embedded in epoxy resin (all from TAAB Laboratories Equipment Ltd., Berks, UK). Ultrathin cross‐sections (60–90 nm) were cut with a Reichert‐ Jung Ultracut ultramicrotome (Leica Microsystems Ltd., Milton Keynes, UK) using a diamond knife. Sections were then mounted on 200 mesh copper grids and stained with “Reynold's Lead citrate” stain (VWR, Leicestershire, UK) for 4 min. Images were obtained using a Philips EM208S Transmission Electron Microscope at 80 KV.