Agar 100 epoxy

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.