Morgagni 268d electron microscope

Cells were fixed with 2.5% glutaraldehyde in phosphate-buffered saline, followed by 2% OsO₄. The thin sections were stained with uranyl acetate and examined using a Morgagni 268D electron microscope (FEI, Hillsboro, OR).

Trophozoites were washed twice with PBS, pH 7.4, fixed with 2.5% (v/v) glutaraldehyde in 0.1 M sodium cacodylate buffer, pH 7.4, for 1 h and post-fixed for 60 min with 1% (w/v) osmium tetroxide. After dehydration with increasing concentrations of ethanol (70, 80, 90 and 100%) and propylene oxide, samples were embedded in Poly/bed^® epoxy resin and polymerized at 60°C for 24 h. Thin sections (60–90 nm) were contrasted with uranyl acetate and lead citrate before being examined in a Philips Morgagni 268 D electron microscope (FEI Company).

Small blocks of motor cortex and striatum were fixed in 300 mM glutaraldehyde (Sigma-Aldrich) in 100 mM cacodylate buffer for 2 h at room temperature (RT), washed in the same buffer and post-fixed in 40 mM osmium tetroxide (Polysciences) in 100 mM cacodylate buffer for 1 h at RT. After rinsing in cacodylate buffer and dehydration in ethanol, the samples were embedded in araldite resin (Durcupan ACM, Sigma-Aldrich). Sections (60 nm thick) were cut using a Leica EM UC6 ultramicrotome, and were stained with uranyl acetate and lead citrate. Sections were examined under an FEI Morgagni 268D electron microscope (FEI Company, The Netherlands) at 70 kV.
This article is part of a special collection ‘A Guide to Using Neuromuscular Disease Models for Basic and Preclinical Studies’, which was launched in a dedicated issue guest edited by Annemieke Aartsma-Rus, Maaike van Putten and James Dowling. See related articles in this collection at http://dmm.biologists.org/collection/neuromuscular.

Immune cells were incubated in a 15 mL tube (Thermo Scientific, USA) (1 μg mL⁻¹ final concentration for 24 h) with gentle agitation, followed by fixing as described above. After intensive washes, cells were post-fixed overnight at 4°C with 1% osmium tetroxide in cacodylate buffer. Samples were washed three times in cacodylate buffer at 4°C and double distilled H₂O, warmed to the RT and embedded into 4% low-melt agarose. Solidified agarose was cut into 1 × 1 mm cubes, dehydrated in ethanol series (25, 50, 75, 90, 96, and 100%), and embedded into the epoxy resin (EMBed-812 Embedding kit; Electron Microscopy Sciences). Ultrathin sections were stained with uranyl acetate and lead citrate, and examined with a FEI Morgagni 268(D) electron microscope (FEI, Czech Republic).

CD14-positive cells were treated with 22.5 pM CyaA toxin or CyaA-AC⁻ toxoid for 5 days as described above and fixed for 2 h with 2% glutaraldehyde–PBS. After three washes with ice-cold PBS, cells were postfixed with 0.5% osmium tetroxide–PBS and incubated overnight at 4°C. Fixed and washed samples were dehydrated with ethanol using a standard procedure. Cells were embedded in epoxy resin (EMBed-812 embedding kit; Electron Microscopy Sciences). Ultrathin sections were visually contrasted using lead citrate and uranyl acetate (54 (link)). Final samples were examined using an FEI Morgagni 268(D) electron microscope (FEI, Brno, Czech Republic) at 80 kV. Digital images were recorded with a MegaViewIII slow-scan camera and processed by AnalySis 3.2 software (Olympus Soft Imaging Solutions GmbH, Münster, Germany) using standard software modules (shading correction, digital contrast enhancement).

Log phase cultures (OD600 0.25 to 0.5) were spotted on a on a 400 mesh copper grid covered with Parlodion film, incubated for 15 min at room temperature, and then fixed in a surface-associated state with 0.1% glutaraldehyde. Preparations were then washed 3 times with water and negatively-stained twice with 1% uranyl acetate. Pictures were taken with a FEI Morgagni 268(D) electron microscope.