Temcam f416

For TEM analysis,
the cells were fixated in 4% paraformaldehyde + 2.5% glutaraldehyde
in PHEM buffer (60 mM PIPES, 25 mM HEPES, 10 mM EGTA, 2 mM MgCl₂, 4% glutaraldehyde, 1% formaldehyde) and incubated for 3
h at room temperature.⁴⁰ In a laboratory
microwave (Pelco BioWave Pro+, Ted Pella, Redding), they were washed
three times in PHEM buffer, incubated using 1% OsO₄ in
PHEM buffer, washed in water, contrasted with 1% uranyl acetate in
water, washed in water and via an ethanol chain,
and acetone-embedded in EponTM. After overnight polymerization, 55
nm-thick sections were cut on an EM UC7 ultramicrotome (Leica, Wetzlar,
Germany) and mounted on 200 MESH copper grids. Post-contrastation
in an automated grid stainer (EM AC20, Leica) and drying, ultrathin
sections were analyzed and micrographed on a Jeol JEM 1400 Plus TEM
equipped with a TemCam-F416 (TVIPS, Gauting, Germany).

For EM, the yeast cells were precultured at 30°C in selective liquid medium containing 2% glucose (1.7 g/liter yeast nitrogen base, 2 g/liter His dropout mix, 5 g/liter ammonium sulfate, and 20 g/liter glucose). The cells were then transferred into selective liquid medium containing 3% glycerol (1.7 g/liter yeast nitrogen base, 2 g/liter His dropout mix, 5 g/liter ammonium sulfate, and 30 g/liter glycerol) and harvested in the logarithmic growth phase. The yeast cells were fixed for 30 min in 2% glutaraldehyde. After several washing steps, the cells were incubated with Zymolyase solution (0.1 mg/ml Zymolyase 100T and 0.5% 2-mercaptoethanol in 1× PBS/10% sorbitol [wt/vol]) for 15 min at room temperature to remove the cell wall. After a second fixation with 1% osmium tetroxide (30 min), the cells were poststained with 1% uranyl acetate for 2 h. After dehydration in a graded ethanol series and propylene oxide, the cells were embedded in epoxide resin (Agar 100; Plano). Ultrathin sections were examined using a transmission electron microscope (CM 120; Philips), and images were taken with a CMOS camera (TemCam F416; TVIPS).

For the visualization of polymerization reactions samples were added to a Formvar (400 mesh) copper-coated grids ionized in BAE 120 Evaporator (Bal-Tec). Samples were adsorbed for 5 min, washed with milli Q water and left for 40 s in 2% uranyl acetate. Visualization was performed in JEM1400 Flash Transmission Electron Microscope (Jeol). Images were taken with a TemCam F416 (TVIPS) camera at a magnification of 20,000X. The extent of tau polymerization was analyzed using the image processing and analysis software Image J (Image J, NIH). Ten random fields from each condition were taken for image analysis. All analyses were performed in a blind manner. Each experiment was repeated at least three times.