Lx 112

Neurons were postfixed in EM fixative buffer, washed in 0.1 M sodium cacodylate buffer (EMS; Hatfield, PA, USA), and postfixed in 1% osmium tetroxide (EMS) for 1  h at 4  °C [31 (link)]. The fixed cells were washed in 0.1 M sodium cacodylate buffer and dehydrated through a graded ethanol series and embedded in LX-112 (Ladd Research Industries; Williston, VT, USA). The sample blocks were sectioned (thickness, 0.5–1 μm) and cut into 90 nm thick sections with an ultramicrotome (Leica EM UC7; Buffalo Grove, IL, USA). The ultra-thin sections were counterstained with uranyl acetate 2% (EMS) and lead citrate. All images were taken with an 80 kV transmission electron microscope (Hitachi, H-7650, V01.07; Tokyo, Japan).

Sections were rinsed in 0.1 m cacodylate buffer and postfixed in 1% OsO₄ and 1.5% K₄Fe(CN) ₆ in 0.1 m cacodylate for 15 min. Another rinse in 0.1 m cacodylate buffer ensued before the sections were dehydrated in an ascending concentration of ethanol and embedded in epoxy resin (LX 112, Ladd Research Industries) according to standard protocols. Sections were flat-embedded between two Aclar sheets at 60˚C overnight. Regions of interest along the dorsoventral axis of MEC LII were carefully cut out and glued with epoxy resin onto polymerized epoxy stubs (Fig. 1b). After polymerization, ultrathin sections of MEC LII were cut using a Leica UC6 Ultramicrotome. Thin sections were collected on 200-mesh thin-bar copper grids and contrasted with 4% uranyl acetate and 1% lead citrate. Sections were inspected with a transmission electron microscope (JEOL JEM-1011) and imaged using a digital Morada camera.

TEM imaging was performed on D. magna samples after exposure to COOH-PS NPs or NH₂-PS NPs. The samples were prefixed with 4% glutaraldehyde in 0.1 M sodium phosphate buffer pH 7.4 for 1 h at 4 °C. After that they were fixed in 1% OsO₄ in 0.1 sodium phosphate buffer for 1 h at 4 °C and were subsequently dehydrated using a gradient of ethanol followed by acetone and LX-112 infiltration followed by embedding in LX-112 resin (Ladd Research Industries, Vermont, OH). Ultrathin sections (50–80 nm) were prepared using a Leica EM UC6 microtome and these were further contrasted with uranyl acetate followed by lead citrate, and finally examined using a Hitachi HT 7700 electron microscope (Hitachi High-Technologies). We used the 2kx2k Veleta CCD camera (Olympus) for image acquisition. TEM analysis was also performed on HT-29 cells. Briefly, cells were detached from the transwells after cell culture and prefixed with 4% glutaraldehyde in 0.1 M sodium phosphate buffer pH 7.4 for 1 h at 4 °C. Thereafter, the procedure described for D. magna samples was followed.

Cells were fixed with 2.5% glutaraldehyde in 0.1 M Sorenson’s buffer (PH 7.2) for at least one hour, then postfixed with 1% OsO4 also in Sorenson’s buffer for one hour. After dehydration cells were embedded in a mixture of Lx-112 (Ladd Research Industries, Inc.) and Embed-812 (EMS, Fortwashington, PA). Thin sections (60 nm) were cut on the MT-Power-Trome XL ultramicrotome. Sections were stained with uranyl acetate and lead citrate and examined under a JEOL JEM-1200 EXII electron microscope. Images were taken on an ORCA-HR digital camera (Hamamatsu) and recorded with the AMT Image Capture Engine.