Ultracut s microtome

Young adult wild-type N2 and ptrn-1(tm5597) animals were prepared as described (Cueva et al., 2012 (link)). Briefly, animals were frozen in an EMPACT2 high-pressure freezer system, and a Leica AFS freeze substitution apparatus (Vienna, Austria) was used to preserve in 2% glutaraldehyde plus 1% osmium tetroxide and embed in Epon/Araldite. A Leica Ultracut S microtome equipped with a diamond knife was used to cut 50-nm serial sections, which were collected on Formvar-coated copper slot grids. The grids were poststained to enhance contrast in 3.5% uranyl acetate (30 s) and Reynold’s lead citrate preparation (3 min). The grids were imaged on a transmission electron microscope (JEOL TEM 1230, Tokyo, Japan), and images were acquired with an 11 megapixel bottom-mounted cooled CCD camera (Orius SC1000, Gatan, Pleasanton, CA).

Overview semi-thin cuts of 1.5 µm thickness were made from the sea slug body part roughly after the pericardium. Semi-thin sections were cut on a Leica UC7 ultramicrotome using a LEICA glass knife and were placed on circular glass cover slips (AGAR SCIENTIFIC, borosilicate, 10 mm diameter, 1½ mm thickness). Histological overviews were documented on an optical light microscope. Thin sections (70 nm) for electron microscopy observations were made on a LEICA ULTRACUT S microtome with a diamond ultra knife (40°) at the electron microscopy facilities of Angers University (SCIAM). The thin-sections were transferred onto formvar-carbon film copper grids and stained for 10 min with uranyless prior observation. The sections were observed with a transmission electron microscope (TEM, Philips 301 CM100, 80 kV) at the electron microscopy facility of Lausanne University (EMF). TEM grids (thin-sections) were transferred on 10 mm aluminum disks with double stick Cu-tape. Before NanoSIMS analysis, both the thin and semi-thin sections were coated with a ca. 15 nm thick gold layer.

The midrib of leaflets 4 or 5 of the L1 leaf was examined by transmission electron microscopy (TEM). Samples were fixed in 2.5% glutaraldehyde–2% paraformaldehyde in 100-mM phosphate buffer, pH 7.2, for 3 h. They were post-fixed overnight at 4 °C with 1% osmium tetroxide, then dehydrated in a graded ethanol series and progressively infiltrated with Epon resin for 48 h. Curing occurred for 24 h at 60 °C. One hundred nanometer-thick sections were cut with an Ultracut S Microtome (Leica) and collected on hexagonal 600 mesh copper grids. Sections were observed at 120KV on a FEI Tecnai G2 Spirit TEM (FEI, Thermo Fisher Scientific, Waltham, MA, USA) equipped with an Eagle 4K digital camera (Fluid + Form by IconnTechs, Wan Chai, Hong Kong). Two plants per genotype were observed for infected plants and one for noninfected. For infected samples, both the transversal and longitudinal sections were analyzed. The number of peroxisomes in the phloem cells was counted per mesh (i.e., region of interest, ROI), one mesh corresponding approximately to 1000 µm². Six–fourteen ROIs, focused on the phloem cells, including phloem parenchyma, phloem perivascular cells and companion cells, were observed per genotype and condition.

SH-SY5Y cells stably expressing full-length human Tau were cultured with 10 μM Congo red in 6-well plates for 2 days and then cultured with 0 or 10 μM myricetin for 2 days, and cells cultured with PBS alone as a negative control. SH-SY5Y cells stably expressing full-length human Tau incubated with PBS were used as a negative control. After prefixation with 3% paraformaldehyde and 1.5% glutaraldehyde in 1 × PBS (pH 7.4), the cells were harvested and postfixed in 1% osmium tetroxide for 1 h using an ice bath; the samples were then dehydrated in graded acetone and embedded in 812 resins. Ultrathin sections of the cells were prepared using a Leica Ultracut S Microtome and negatively stained using 2% uranyl acetate and lead citrate. The doubly stained ultrathin sections of cells were examined using a JEM-1400 Plus transmission electron microscope (JEOL) operating at 80 kV.

SH-SY5Y cells and HEK-293T cells were cultured in 6-well plates in the minimum essential medium for 1 day and then cultured with 0 or 10 μM SOD1 fibril seeds for 3 days, and cells cultured with 20 mM Tris-HCl buffer (pH 7.4) containing 5 mM TCEP as a negative control. After prefixation with 3% paraformaldehyde and 1.5% glutaraldehyde in 1× PBS (pH 7.4), the cells were harvested and postfixed in 1% osmium tetroxide for 1 h using an ice bath; the samples were then dehydrated in graded acetone and embedded in 812 resins. Ultrathin sections of the cells were prepared using a Leica Ultracut S Microtome and negatively stained using 2% uranyl acetate and lead citrate. The doubly stained ultrathin sections of cells were examined using a JEM-1400 Plus transmission electron microscope (JEOL) operating at 100 kV. The TEM images were analyzed by using Origin Pro software version 8.0724 (Origin Laboratory), and p values were determined using two-sided Student’s t test. All experiments were further confirmed by biological repeats.

Mature worm couples were treated for 48 hours with 5 μM PHX or DMSO. Next male parasites were fixed for 24 hours at 4°C with a mixture of 2% (w/v) paraformaldehyde, 2.5% (w/v) glutaraldehyde (TAAB) in 0.1 M phosphate buffer, pH 7.4 and post-fixed with 1% (w/v) OsO₄ supplemented with 1.5% (w/v) potassium ferrocyanide for 2h on ice. Subsequently samples were dehydrated in ethanol and infiltrated with propylene oxide (Agar) / Epon (Agar) (1:1) followed by epon embedding (48h at 60°C). Ultrathin sections (50 nm) were cut with an Ultracut S microtome (Leica), counter-stained with lead citrate and observed with a transmission electron microscope Jeol 1010. Images were obtained using a Gatan MSC 791 CCD camera (Gatan).