Procure 812

Samples were incubated in propylene oxide (ProSciTech, 2×5 min) and embedded in resin. Embedding consisted of a 2 h incubation in 1:1 mix of propylene oxide and resin (Procure 812, ProSciTech); 10 min incubation at 60°C in resin, followed by overnight incubation at room temperature in the dark in resin; and finally embedding in resin at 60°C for at least 24 h to allow curing.

After 42 days of feeding, one fish from each tank was randomly euthanized with AQUI-S at 175 mg/L to excise liver, muscle, gill, and intestine for histological and TEM evaluation in response to test diets. For histological analysis, samples of all organs were fixed immediately in 10% buffered formalin, subsequently dehydrated with series of ethanol, infiltrated in xylene, and embedded in paraffin wax, as per standard histological protocols. Section of approximately 5 μm thickness was stained with Periodic Acid-Schiff (PAS) and digitally photographed under a light microscope (BX40F4, Olympus, Tokyo, Japan).
For TEM analysis, freshly collected intestinal samples washed in 2.5% glutaraldehyde buffered in 1x PBS at pH 7.4 before performing secondary fixation in 1% OsO4 (80 W 2 min on, 2 min off, 2 min on), dehydrating in ethanol (50, 70, 95 and 100% at 250 W, 40 seach) and infiltrating finally with epoxy resin in acetone (Procure 812, Proscitech) (1:3, 1:1, 3:1ratios at 250 W, 3 min each). Samples were processed as described in the earlier study in our lab [16 (link)] and screened a LaB6 TEM (JEOL2100, Japan) at 120 kV. The electron micrographs obtained from TEM analysis at 30,000 magnification were analysed using ImageJ (National Institute of Health, USA) to determine microvilli length and diameter.

Scanning electron microscopy (SEM) and transmission electron microscopy (TEM) was performed at the Manawatu Microscopy and Imaging Centre, Massey University, Palmerston North, New Zealand. SEM was performed on an aliquot of sample 3 (see section on Quinella-enriched samples, above) fixed to a Formvar grid (Sigma-Aldrich, St. Louis, MO, USA), stained with 2% (w/v) uranyl acetate and examined with a FEI Quanta 200 scanning electron microscope (Philips Electron Optics, Eindhoven, The Netherlands). Part of sample 3 was prepared for TEM by washing the cell pellet three times in sterile water, resuspending in modified Karnovsky’s fixative (2% [w/v] paraformaldehyde and 3% [w/v] glutaraldehyde in 0.1 M sodium phosphate buffer, pH 7.2) for embedding in resin (Procure 812; ProSciTech, Qld, Australia), and thin sections made using an EM UC7 ultra-microtome (Leica Microsystems, Wetzlar, Germany). TEM used a Tecnai G2 Biotwin transmission electron microscope (FEI, Hillsboro, OR, USA). Electron microscopy used XT Microscope Control and TUI version 4.5 software (FEI).