Hexamethyldisilazane

Samples were fixed overnight in 4% PFA, dehydrated in graded ethanols, incubated for 20 min in 50% hexamethyldisilazane (Sigma-Aldrich Co.) in ethanol followed by three changes of 100% hexamethyldisilazane, air-dried overnight, mounted on stubs and sputter coated with gold palladium. Specimens were observed and photographed using a Quanta 250 scanning electron microscope (FEI, Hillsboro, OR, USA) at 10 kV accelerating voltage.

Fe(0) particles were collected from cultures when succinate production plateaued and were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer for 12 h at 4°C. They were then washed 3 times in 0.1 M phosphate buffer at 4°C for 10 min and then dehydrated in successive ethanol-water mixtures of 35%, 50%, 70%, 80%, 90%, 95%, and 100% for 10 min. The 100% ethanol step was repeated 3 times. Samples were further dehydrated in a 50% hexamethyldisilazane (Sigma-Aldrich, St. Louis, MO, USA) ethanol solution by gentle mixing for 3 min at room temperature, immersed in pure hexamethyldisilazane for 3 min at room temperature, and dried with a stream of high-purity nitrogen for 30 min. Scanning electron microscopy was conducted with an ultrahigh-resolution field emission scanning electron microscope (FEI Magellan 400; Nanolab Technologies, CA, USA).

Juveniles grown in 50% CS in RPMI were fixed in 4% glutaraldehyde (Sigma-Aldrich), for 4 h at 4°C. Following this, juveniles were washed in 0.1 M sodium cacodylate buffer (0.1 M sodium cacodylate (Sigma-Aldrich) buffer (pH 7.4), containing 3% sucrose (Sigma-Aldrich)) overnight (~16 h) at 4°C. Juveniles were then stained in 1% OsO₄ (90 min, 4°C), followed by three 15 min washes in H₂O at RT. Juveniles were then washed twice in 70% ethanol for 30 min, twice in 90% ethanol for 20 min and twice in 100% ethanol for 5 min (all at RT). Juveniles were covered with 200 μl hexamethyldisilazane (Sigma-Aldrich) and after 5 min this was removed and 200 μl of fresh hexamethyldisilazane was added and allowed to evaporate overnight (~16 h, RT). Juveniles were then transferred onto stubs and sputter coated for 5 min using a Polaron E5100 Series II before viewing under a FEI Quanta 200 scanning electron microscope. Image J software was used to measure the length of ten spines within the first three rings surrounding the oral suckers of 2–3 juveniles at each week of culture following excystment.

Tongue samples were fixed in 4% PFA overnight and dehydrated in a graded series of ethanol concentrations. Dehydrated samples were then incubated in 50% hexamethyldisilazane (Sigma-Aldrich, St. Louis, USA) for 20 min, followed by three solvent changes to 100% hexamethyldisilazane. After air-drying overnight, the samples were sputter-coated with gold-palladium. Specimens were examined and photographed using a SEM^{49 (link)}.

The Caco-2/Raji B and Caco-2/HT29-MTX co-cultures were exposed to cell culture medium (control) or 12.68 Cu µg/cm² of CuO NMs (500 µl/well) for 24 h at 37 °C. After 24 h, the inserts were washed with PBS twice, fixed with 5% glutaraldehyde (in 0.1 M sodium cacodylate) for 2 h at 4 °C. The cells were washed three times with 0.1 M sodium cacodylate followed by dehydration in graded ethanol (25, 50, 70, 80 and 90%) for 10 min in each ethanol grade at RT. The cells were further dehydrated in 100% ethanol three times for 15 min each then, submerged in 2:1 fresh solution of hexamethyldisilazane (Sigma, Poole):100% ethanol. The glass coverslips containing the cells were dried in 100% hexamethyldisilazane (Sigma, Poole) and mounted on SEM specimen stubs (Aluminium, 12.5 mm diameter, 3.2 × 6 mm pin Agar Scientific UK). The polycarbonate membranes were carefully excised and mounted on SEM specimen stubs (Aluminium, 12.5 mm diameter, 3.2 × 6 mm pin Agar Scientific UK). Then, specimens were coated with gold and examined with SEM. More than 5 views were imaged, and representative images presented.

After thrombin generation was determined in whole blood, the clots were prepared for visualization by SEM. The clots were fixated by adding 2.5% glutaraldehyde (grade I, Sigma Aldrich, St. Louis, Missouri) in PBS (Sorensen’s, pH 7.2) (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hour at room temperature and then placing it at 4°C overnight. The following day, the glutaraldehyde solution was removed and the samples were repeatedly (5x) washed with PBS. As a secondary fixation, the samples were placed in osmiumtetroxide (OsO₄, 1%) diluted in sodium cacodylate (200 mM, pH 7.4) (Electron Microscopy Sciences, Hatfield, PA, USA) for 1 hour at room temperature. Consecutively, the clots were dehydrated in ethanol (30%, 50%, 70%, 90% and 3 times at 100%) for 3 minutes. The samples were then treated with a hexamethyldisilazane/ethanol solution for 3 minutes and in hexamethyldisilazane (Sigma Aldrich, St. Louis, MO, USA) for 10 minutes. The samples were removed from the wells, left to dry and coated with gold. Analysis was performed on a desktop SEM (Phenom-World, Eindhoven, the Netherlands).

The organoids were analysed by brightfield and scanning electron microscopy (SEM). The samples were analysed on days 1, 7, 14, and 21 during differentiation. For SEM, the samples were cut to 1 mm diameter, fixed in 4% glutaraldehyde (Sigma-Aldrich), and then gradually dehydrated through an ethanol series, using sequentially higher concentrations of ethanol (70%, 80%, 90%, 95%, and 100%) for 10 min each. Finally, samples were further dehydrated in 50% hexamethyldisilazane (Sigma-Aldrich) diluted with ethanol for 10 min, followed by full immersion in absolute hexamethyldisilazane, and left to evaporate overnight in a fume cupboard. Control scaffold particles (no cells) were also incubated for 7 days in organoid medium, at 37°C under 5% CO₂ in air, before being fixed and dehydrated. SEM was carried out using a Hitachi S-4800 II SEM with an accelerating voltage of 1 kV at 110x and 5000x. Samples were mounted onto SEM stubs using a double-coated carbon conductive tape (Acros Organics, supplied by Fisher Scientific).
Cell viability was also assessed after 14 and 21 days using Live/Dead assay kit (Life Technologies, Paisley, UK), in which cells were stained with calcein acetoxymethyl (0.1 μg/ml) and propidium iodide (1 μg/ml) and viewed by fluorescence microscopy.