1200ex electron microscope

Recombinant human and mouse wild-type α-synuclein and fibrils were prepared as described previously [33 , 57 (link)]. Briefly, purified α-synuclein (7 – 10 mg/ml) was incubated at 37 °C in a shaking incubator at 200 rpm in 30 mM Tris–HCl, pH 7.5, containing 0.1% NaN₃, for 72 h. α-Synuclein fibrils were pelleted by spinning the assembly mixtures at 113,000 xg for 20 min, resuspended in 30 mM Tris–HCl buffer (pH 7.5), and sonicated for 3 min (Biomic 7040 Ultrasonic Processor, Seiko). The protein concentrations were determined by HPLC. Samples were run on gradient 12% polyacrylamide gels and stained with Coomassie Brilliant Blue (CBB), or electrophoretically transferred to PVDF membranes. For immunoblotting, membranes were incubated with 3% gelatin (Wako) for 10 min at 37 °C, followed by overnight incubation at room temperature with primary antibodies. Next, the membranes were incubated for 1 hr at room temperature with biotinylated anti-rabbit or mouse IgG (Vector Lab), then incubated for 30 min with avidin-horseradish peroxidase (Vector Lab), and the reaction product was visualized by using 0.1% 3,3-diaminobenzidine (DAB) and 0.2 mg/ml NiCl₂ as the chromogen. For electron microscopy, samples were placed on collodion-coated 300-mesh copper grids, stained with 2% (v/v) phosphotungstate, and examined with a JEOL 1200EX electron microscope.

For basal complex development stage analysis, HFF infected cells were fixed in 4% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 and processed for routine electron microscopy (Ferguson et al., 1999 (link)). Briefly, cells were post-fixed in osmium tetroxide and treated with uranyl acetate prior to dehydration in ethanol, treatment with propylene oxide, and embedding in Spurr’s epoxy resin. Thin sections were stained with uranyl acetate and lead citrate prior to examination with a JEOL 1200EX electron microscope.
Basal complex mutant infected cells were prepared for ultrastructural observations by fixation in 2.5% glutaraldehyde in 0.1 mM sodium cacodylate (EMS) and processed as described (Coppens and Joiner, 2003 (link)). Ultrathin sections were stained before examination with a Philips CM120 EM (Eindhoven, the Netherlands) under 80 kV.

Extracellular parasites (±Tf-LPA/NGP, see NGP uptake above) were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer, pH 7.4, after the indicated incubation. Samples were processed for routine electron microscopy as described previously [69 (link)] and examined in a JEOL 1200EX electron microscope.

Dictyostelium species were grown in association with Klebsiella aerogenes on 1/5^th SM agar for robust species and either LP, 1/3^rd LP or non-nutrient agar plates with charcoal for delicate or very delicate species, respectively^{8 (link)}, and left to develop into fruiting bodies after bacteria were cleared. Mature spores were harvested from 4 day-old fruiting bodies. Cysts were induced by incubating cells, freshly harvested from growth plates, for 3–5 days in darkness submerged in PB or 250 mM sorbitol in PB, depending on the species^{8 (link)}. Cysts were harvested when fully encysted, as established by Calcofluor staining of the cellulose wall. Spores and cysts were pelleted and fixed by submersion in 0.83% glutaraldehyde, 0.67% OsO4, 2.5% sucrose in 0.1 M sodium cacodylate pH 7.4 as described previously^{25 (link)}. Dehydration, embedding, and sectioning used conventional techniques. Images were captured on Ditabis imaging plates or a SIS Megaview III camera using a JEOL 1200EX electron microscope. For each species, 10 images of complete spore- and cyst sections where collected at magnifications between 10 and 20 K, as well as close-ups of regions of the cell wall at 60 K. Sections were selected that were not obviously oblique, included a fair-sized nucleus and went through spores longitudinally, to ensure that they passed approximately through the centre of the cell.

Macrophage cells were cultured for 3 days in 75-cm² flasks prior to be incubated for 24 h with native oocysts at ratio 1:1. Then, cells were washed twice with 0.1 M phosphate buffer and gently detached with a cell scrapper. Following centrifugation (400 g, 10 min), cells were resuspended and fixed in 2.5% glutaraldehyde in 0.1 M phosphate buffer. The samples were post-fixed in osmium tetroxide, dehydrated in ethanol, treated with propylene oxide and embedded in Spurr’s epoxy resin. Thin sections were cut and stained with uranyl acetate and lead citrate prior to examination in a Jeol 1200EX electron microscope.