Reichert ultracut e microtome

Upon receiving the samples, tissue blocks were trimmed and left in 4% formalin for a period of 10 days. Demineralization was performed by leaving the tissue blocks in 10% ethylenediaminetetraacetic acid (EDTA) for 5 weeks. Tissue blocks were divided into 3 sections and embedded in paraffin, methylmethacrylate (MMA), or in acrylic resin (LR White, Sigma–Aldrich, St. Louis, MO, USA). Paraffin sections were cut at 5 μm, while MMA-embedded tissues were cut with a diamond-coated disc and ground to a thickness of 100 μm. LR White sections were cut at 1 μm with a diamond knife on a Reichert Ultracut E microtome (Leica Microsystems, Wetzlar, Germany). MMA-embedded sections were stained with basic fuchsin and toluidine blue, and LR White-embedded sections were double stained with toluidine blue and basic fuchsin. Paraffin embedded sections were stained with hematoxylin/eosin, Giemsa, or Masson’s trichrome. Digital photography was performed using a digital camera (AxioCam MRc, Carl Zeiss, Oberkochen, Germany) connected to a microscope (Axio Imager M2, Carl Zeiss, Oberkochen, Germany).

CuO NPs have an average particle size of 40 nm, a specific surface area of 131 m²/g, and a purity of 99.9% according to the product information from the manufacturer (Beijing Nachen Technology Co., Beijing, China). CuO NP powders were embedded in the resin and heated overnight at 70 °C, then were sliced into 90 nm with a Reichert Ultra Cut E microtome (Leica Microsystems AG, Wetzlar, Germany) and mounted on a nickel grid. The morphology and size of CuO NPs were observed using a TEM (Hitachi, H-7650, Tokyo, Japan) with an operating voltage of 60 kV.

For standard (2D) electron microscopy imaging, 80-nm sections were cut from the embedded sample blocks on a Reichert Ultracut-E microtome (Leica Microsystems, Vienna) using a diamond knife (Diatome, Biel, Switzerland) and were collected on uncoated 100- and 200- mesh copper grids (hexagonal or square meshes; Ted Pella, Redding, CA, USA). The sections were post-stained with 1% uranyl acetate (Canemco & Marivac, Gore, QC, Canada; pH not recorded) and Reynolds lead citrate (Fisher Scientific Company, USA; pH not recorded) for 12 and 6 minutes, respectively. Electron micrographs at various magnifications were obtained with a Hitachi 7600 transmission electron microscope (Hitachi High Technologies, Japan) at 80 kV.
Lysosomes in these images were identified according to their well established appearance and features: they are bound by a single lipid-bilayer membrane, with a granular, more or less uniform luminal matrix that is more electron dense than the surrounding cytosol. Secondary lysosomes may also contain less granular structures within the finer matrix. Moreover, lysosomes are normally distinguishable from endosomes by their larger size, hence we set a threshold “diameter” of > 200 nm for acceptance of a lysosome, below which all vesicles were excluded.