208s electron microscope

Fragments of xenograft tissue were fixed in commercially prepared fixative (Electron Microscopy Sciences, Hatfield PA) containing 2.5% glutaraldehyde and 2% formaldehyde in 0.1 M sodium cacodylate buffer. They were then postfixed in 2% OsO₄, dehydrated in a graded ethanol series followed by propylene oxide, and embedded in Epon. Ultrathin (90 nm) sections on copper grids were stained with 0.2% lead citrate and 1% uranyl acetate and examined with a Philips 208S electron microscope at 80 kV.

Cells were fixed with 2% glutaraldehyde, 2% paraformaldehyde (PFA) in 0.1 M PBS for transmission EM and 4% PFA in in 0.1 M PBS for IEM. For EM, the cells were post-fixed in 1% OsO4, washed in distilled water for 3 times, stained with 1% uranyl acetate in 50% ethanol, dehydrated with 70%, 80%, 95% and 100% ethanol then propylene oxide, infiltrated and embedded in Epon 812 (Polysciences, Warrington, PA, USA). For IEM, cells were exposed in 30%, 50%, 70% and 90% ethanol then 90% ethanol-LR White (1:1), 90% ethanol-LR White (1:2), and infiltrated and embedded in pure LR White. Ultrathin sections were cut from the Epon 812 or LR White embedded samples with a Leica Ultramicrotome. Tissue sections on EM grids were subjected to immunogold labeling and counterstained with uranyl acetate and lead citrate. Results were examined and photographed with a Philips 208S electron microscope.

His-tagged full-length tau, tau 1–255, 1–368, 256–368, 256–441, and 368–441 was purified from E. coli using His bind purification kit (Calbiochem). The purified tau fragments were induced to aggregate as described previously ^{43 (link)}. Briefly, purified tau fragments (50 μM) were incubated in PBS (pH 7.4) containing 12.5 μM heparin, 2 mM DTT and a protease inhibitor cocktail. The samples were incubated with 50 μM thioflavin S for 45 min in the dark at room temperature. PHF formation was quantified by measuring the fluorescence with an excitation at 440 nm and an emission wavelength of 510 nm. The overall appearance of the PHFs was visualized by negative stain electron microscopy. Briefly, the reaction samples was adsorbed onto carbon/formvar-coated 400 mesh copper grids (EM Sciences) for 30 s, and stained with 2% uranyl acetate for 30 s. Excess liquid in the sample was wicked using filter paper. The grids were examined with a Philips 208S electron microscope (Philips, Hillsboro, OR). The remaining solutions of aggregated tau were centrifuged at 100,000 g for 30 min to separate aggregated tau pallet and non-aggregated tau supernatant, and analyzed by Western blot.

Tau assembly reactions were diluted 1:2 in Buffer A (10 mM HEPES pH 7.4, 50 mM NaCl, 1.5 mM MgCl₂, 0.5 mM DTT, 2.5 mM EGTA, 0.1 mM EDTA), and 10 ml were absorbed onto carbon/formvar-coated 400 mesh copper grids (Electron Microscopy Sciences, Hatfield, PA) for 45 s, then washed twice for 15 s in Buffer A to remove remaining guanidine hydrochloride. Tau fibrils were counterstained with 2% uranyl acetate (Electron Microscopy Sciences, Hatfield, PA) for 45 seconds and examined using a Philips 208S electron microscope (Philips, Hillsboro, OR), with images collected at 5000 × magnification. For Immuno-EM staining, the anti-tau primary antibody was detected with a 6 nm-gold particle coupled secondary antibody, and anti-Nup98 was detected using a secondary antibody conjugated with 12 nm gold particles.