Orius sc200b

Extracted tau filaments were deposited on glow-discharged 400 mesh formvar/carbon film-coated copper grids (EM Sciences CF400-Cu) for 40 s, blocked for 10 min with PBS + 0.1% gelatin, and incubated with primary antibody (1:50) in blocking buffer, essentially as described [20 (link)]. Primary antibodies were BR136 (raised against residues 244–257) [12 (link)], Anti-4R (raised against residues 275–291, with D279) [10 (link)], BR135 (raised against residues 323–335) [21 (link)], and TauC4 (raised against residues 354–369) [38 (link)]. Where stated, grids were incubated for 5 min with 0.4 mg/ml pronase (Sigma) in PBS at room temperature and washed with blocking buffer, prior to blocking. Following incubation with primary antibodies, grids were washed with blocking buffer and incubated with 10 nm gold-conjugated anti-rabbit IgG (Sigma) diluted 1:20 in blocking buffer. The grids were then washed with water and stained with 2% uranyl acetate for 40 s. Images were acquired at 11,000 × and 15,000 ×, with a defocus value of -1.4 μm with Gatan Orius SC200B or Gatan Ultrascan 1,000 CP CCD detectors using a Tecnai G2 Spirit at 120 kV. To distinguish non-specific background levels of isolated gold beads from specific labelling of the filaments, we only considered filaments with more than six gold beads to be positively labelled.

All the experiments were performed with a Tecnai Femto ultrafast electron microscope (FEI Company) operated at 200 kV in both thermionic and photoelectron modes. In both modalities, a truncated, 150-μm flat LaB₆ cathode (Applied Physics Technologies) was used. To capture the greatest number of photoelectrons at the relatively low repetition rates used here59 (link), a custom 1,250-μm condenser aperture was used for all the experiments. For all the bright-field experiments, a 40-μm objective aperture was used. For all the selected-area diffraction experiments, a 200-μm projection aperture was used, which collected electrons passing through a 20-μm² area. This same aperture can be seen in the bright-field-imaging Supplementary Videos 1 and 5. The images were recorded with a Gatan Orius SC200B 2,048 × 2,048 CCD camera and with integration times ranging from 13 to 20 s per frame. On the basis of the total electron counts (approximately (1 × 10⁸) to (5 × 10⁸)) acquired with the beam focused to the size of the CCD chip for a given exposure time and repetition rate, it is estimated that 200 to 1,000 electrons per pulse were used for image formation.