Poly bed

To determine the embryonic origin of the cells expressing specific Hau-slit and Hau-robo1, teloblasts or proteloblasts of embryos in stages 5 and 6 were injected with a fluorescently labeled, fixable dextran lineage tracer, fluorescein-conjugated dextran amine (FDA), as previously described [41 (link)]. Injected embryos were cultured in Helobdella triserialis saline (HTR) at 23 °C to the desired embryonic stage, then fixed, and processed by FWMISH as described above. After FWMISH, the embryos were dehydrated in ethanol and propylene oxide, followed by infiltration with plastic embedding medium (PolyBed, Polysciences, Inc.). Then embryos were sectioned by using a glass knife on a microtome (MT-2B; Sorvall, Newtown, CT, USA) or hand cut by razor blade into 0.1 mm sections. Sections were imaged on a Leica compound microscope to obtain combined fluorescence images (Leica, Wetzlar, HE, Germany).

Three quadriceps and three hearts per group were dissected and fixed in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde (Electron Microscopy Science, Hatfield, PA, USA), for 2 h at room temperature. Samples were postfixed in osmium tetroxide for 2 h and 1% uranyl acetate for 2 h. Samples were next dehydrated through a graded ethanol series and propylene oxide and embedded in epoxy resin (Poly-Bed; Polysciences, Inc., Warrington, PA, USA) overnight at 42 °C and 2 days at 60 °C. Ultrathin sections (50 nm) were counterstained with 5% uranyl acetate and lead citrate and observed with transmission electron microscope (TEM) HT7880, Hitachi, Japan.

For TEM, LLC-MK₂ cultures were infected with tachyzoites as described above and then treated with 1 and 5 μM Et-Cipro and Adam-Cipro for 24 and 48 h, 20 μM Cipro for 48 and 72h or left untreated (control). After treatment, cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4), and post-fixed for 45 min (and in the dark) in 1% osmium tetroxide, 1.25% potassium ferrocyanide and 5 mM CaCl₂, in 0.1 M sodium cacodylate buffer (pH 7.4). Samples were dehydrated in acetone solutions of increasing concentrations (30–100%) and embedded in PolyBed (Polyscience Inc., Warrington, PA, USA). Ultrathin sections were stained with uranyl acetate and lead citrate, and then observed in a Zeiss 900 Electron Microscope (Carl Zeiss, Inc.) or in a Jeol 1200 EX electron microscope (Jeol LTD, Tokyo, Japan).

A1 cells were seeded in glass chamberslides (Lab-Tek, Nunc, 177380) for 48 h at 37 °C and treated for further 48 h with serum-free medium, PrP90-231 (1 µM), and PrP90-231 (1µM) with rapamycin (10 nM). Cells were washed with 0.1 M cacodylate buffer in distilled water and fixed in the same buffer containing 2.5% glutaraldehyde for 1 h at r.t. Cells were postfixed in 1% osmium tetroxide for 10 min and 1% uranyl acetate for 1 h, dehydrated through a graded ethanol series embedded in epoxy resin (Poly-Bed; Polysciences, Inc, Washington, PA, USA) overnight at 60 °C. Ultrathin sections (50 nm) were observed with a CM10 (Philips, Eindhoven, The Nederlands) without additional staining. Digital images were taken with Megaview 3 CCd camera and iTEM software and processed with Adobe Photoshop CS5.

2D neuronal culture cells (DIV 57) were washed out in 0.1 M cacodylate buffer and fixed in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde (Electron Microscopy Science, Hatfield, PA, United States), for 1 h at room temperature. The cells were postfixed in 1% osmium tetroxide for 2 h and 1% aqueous uranyl acetate for 1 h. Subsequently, samples were dehydrated through a graded ethanol series and flat embedded in epoxy resin (Poly-Bed; Polysciences, Inc., Warrington, PA) for 24 h at 60C. Ultrathin sections (50 nm) were cut parallel to the substrate and counterstained with 5% uranyl acetate in 50% ethanol. Electron micrographs were acquired as single snapshots and MIA (multiple image alignment) at Hitachi 7,800 120Kv electron microscope (Hitachi, Tokyo, Japan) equipped with a Megaview III digital camera and Radius software (EMSIS, Muenster, Germany).

Primary neuronal cultures from PS19 mice expressing P301S mutant human tau and cultured NSC-34 cells transfected with vehicle or human TDP-43 expressing plasmid were fixed, 40 h after transfection, with 2.5% glutaraldehyde (Electron Microscopy Science) in 0.1 M cacodylate buffer for 1 h at room temperature, post-fixed in 1% OsO₄ for 1 h, 1% tannic acid for 30 min and en bloc stained with 1% uranyl acetate for another hour. Then samples were dehydrated through a graded ethanol series and flat embedded in epoxy resin (Poly-Bed; Polysciences, Inc.) for 24 h at 60 °C. Ultrathin sectioning (50 nm) was performed with Leica ultramicrotomes (Reichert Ultracut, Leica microsystems). Flat-embedded cells were cut parallel to the substrate and counterstained with 5% uranyl acetate in 50% ethanol. Ultrastructural analysis was performed with a Hitachi 7800 120 kV electron microscope (Hitachi) operating at 100 kV using a Megaview G3 digital camera and Radius software (EMSIS). Electron micrographs were taken using the Multiple Image Alignment (MIA) montage and screenshot tools.

Cells grown on glass chamber slides (Lab-Tek, Nunc, 177380) were washed in 0.1 M cacodylate buffer and fixed in 0.1 M cacodylate buffer containing 2.5% glutaraldehyde for 1 h at room temperature. Cells were post-fixed in osmium tetroxide for 2 h and 1% uranyl acetate for 1 h. Samples were dehydrated through a graded ethanol series and flat embedded in resin (Poly-Bed; Polysciences, Inc., Warrington, PA, USA) for 24 h at 60°C. Ultrathin sections (50 nm) were cut parallel to the substrate until reaching the apical surface, stained with 5% uranyl acetate in 50% ethanol, and observed with CM10 electron microscope (Philips, Eindhoven, the Netherlands), images were taken with a Megaview 3 camera. Mitochondrial number and size were assessed in 12 randomly taken images at 25k magnification for each treatment. The mitochondrial length (major axis) and width (minor axis) were measured with iTEM software package (Olympus-SYS). Statistical analysis for mitochondrial diameter was performed using the Mann–Whitney test. The total number of mitochondria and mitochondrial cristae was manually scored. Statistical analysis was performed with Student’s t-test.