Tecnai g2 f20 s twin transmission electron microscope

The end-point products of aggregated samples were placed on glow-discharged, 400-mesh Formvar carbon-coated copper grids (EMS Inc., Hatfield, PA, USA) for 3 min (Aβ or α-synuclein) or for 1 min (TrxHttex1-polyQ), rinsed, and negatively stained with 2% uranyl acetate (UA). Samples were examined in Hitachi H-7000 TEM (Hitachi Inc., Tokyo, Japan) at an accelerating voltage of 75 kV. For immunogold staining, the incubated TrxHttex1-39Q (50 μM) with and without equimolar SERF1a samples were dropped on the grids for 10 min and rinsed. After air-drying, the grids were then probed with primary SERF1a antibody (1:100; monoclonal antibody) and the 10 nm gold-conjugated secondary anti-mouse IgG antibody (1:500; abcam). The grids were finally stained with 1% UA. The samples were observed with a FEI Tecnai G2 F20 S-TWIN transmission electron microscope at 120 kV with TEM user interface and DigitalMicrograph software.

After indicated treatments, cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer and stored at 4°C until embedding. Then, cells were postfixed with 1% OsO₄ in 0.1 M cacodylate buffer (pH 7.2), containing 0.1% CaCl₂ for 1 h at 4°C. After rinsing with cold distilled water, cells were dehydrated through a graded series of ethanol (30%–100%). Samples were embedded in EMbed-812 (EMS, 14120), and after polymerization of the resin at 60°C for 36 h, serial sections were cut using an ultramicrotome (Leica, Germany) and mounted on formvar-coated slot grids (EMS, GA300-Cu). Sections were stained with 4% uranyl acetate and lead citrate and examined under a Tecnai G2 F20 S-TWIN transmission electron microscope (FEI, American).