Cm120 biotwin tem

Copper grids (300-mesh) coated with a Formvar/carbon support film (Agar Scientific Ltd, Stansted, UK) were prepared by glow discharge in an Emitech K350G system (Emitech LTD, Nicosia, Cyprus). Nanocomplex preparations were applied to grids as a droplet then after 5 seconds, the grid was dried by blotting with filter paper. The sample was then negatively stained with 1% uranyl acetate for 2–3 seconds, before blotting with filter paper and air-dried. Imaging was performed with a Philips CM120 BioTwin TEM and operated at an accelerating voltage of 120 kV. The images were captured using an AMT 5 MP digital TEM camera (Deben UK Limited, Suffolk, UK).

Fifth-instar larvae of Stenopsyche marmorata (Fig. 1 insert) were collected from Sagami River. The silk glands were dissected and immediately fixed in a prefixative solution of 2% paraformaldehyde and 2.5% glutaraldehyde for 6 h at room temperature. Divided pieces of the silk glands were repeatedly rinsed with 0.1 M cacodylate buffer and then postfixed in 1.5% OsO₄ solution for 2 h at room temperature. After dehydration with a graded series of ethanol, the pieces were embedded in Epon 812 (TAAB, Berkshire, UK). For optical microscopic observation, semi-thin sections (0.75 μm) were cut with a glass knife and stained with azure B. Ultrathin sections (70–90 nm) were cut on a Reichert OMU 3 with a 45° diamond knife (Diatome, Biel, Switzerland) and stained with a solution containing lead acetate, lead citrate, and lead nitrate. Stained sections were examined with a Phillips CM120 Biotwin TEM operating at 80 kV accelerating voltage.

Nanocomplexes were prepared as described above and were applied onto a glow‐discharged 300‐mesh copper grid coated with a Formvar/carbon support film (Agar Scientific). The grid was dried by blotting with filter paper then negatively stained with 1% w/v uranyl acetate before blotting with filter paper and air‐dried. Imaging was performed under a Philips CM120 BioTwin TEM, operated at an accelerating voltage of 120 KV.

Nanoparticles were applied to a copper grid (300-mesh) coated with Formvar/carbon support film (Agar Scientific, Stanstead, UK). They were then incubated for 3 min at room temperature. The samples were negatively stained with uranyl acetate (1%) for 15 s, followed by air drying and blotting on filter paper. A Philips CM120 BioTwin TEM was used for imaging (accelerating voltage 120kV). The images were captured using an AMT 5MP digital TEM camera (Deben UK, Suffolk, UK).

Tissue was prepared for TEM as previously described (Karttunen et al., 2017 (link)). Briefly, zebrafish embryos were terminally anaesthetised in tricaine and incubated, with microwave stimulation, first in primary fixative (4% paraformaldehyde + 2% glutaraldehyde in 0.1M sodium cacodylate buffer) and then in secondary fixative (2% osmium tetroxide in 0.1M sodium cacodylate/imidazole buffer). Samples were then stained en bloc with a saturated uranyl acetate solution and dehydrated in an ethanol series and acetone, both with microwave stimulation. Samples were embedded in EMbed-812 resin (Electron Microscopy Sciences) and sectioned using a Reichert Jung Ultracut Microtome. Sections were cut at comparable somite levels by inspection of blocks under a dissection microscope and stained in uranyl acetate and Sato lead stain. TEM images were taken with a Phillips CM120 Biotwin TEM. The Photomerge tool in Adobe Photoshop was used to automate image registration and tiling. To assess axon diameter and g-ratio, axonal areas were measured in ImageJ with and without their myelin sheath, and diameters (d_axon and d_axon+myelin, respectively) calculated from the obtained values. G-ratios were calculated by dividing axon diameter, d_axon, by the axon + myelin diameter, d_axon+myelin.