Jem 1400 plus transmission electron microscope

Mice were anesthetized and perfused in Karnovsky’s fixative solution (2% formaldehyde and 2.5% glutaraldehyde in 0.1 M Sodium Cacodylate buffer, pH 7.4). Mouse brains were removed and 100 μm coronal sections were obtained using a vibratome (Leica Biosystems, IL, USA). Brain sections were fixed in Karnovsky’s fixative solution for 2 h at room temperature and then transferred to 4 °C for an additional 24 h. Sections were washed four times with 0.1 M sodium cacodylate buffer (pH 7.3) and incubated for 1 h in 1% osmium tetroxide, 1.5% potassium ferricyanide in sodium cacodylate. Sections were then washed 4 times in the same buffer; dehydrated with graded series of ethanol solutions (30, 50, 70, 80, 90, 95%) for 10 min each; then in 100% ethanol 3 times for 20 min each; followed by two changes of propylene oxide. Brain sections were infiltrated with series of epoxy resin, (25, 50, 75, 100%) for 24 h each and polymerized in the oven at 60 °C for 48 h. The blocks were sectioned by an ultramicrotome (Ultracut E, Riechert-Jung, Ontario, Canada) and sections of 80 nm were stained with uranyl acetate and lead citrate. Sections were observed using a Jeol JEM 1400 Plus Transmission Electron Microscope and pictures were taken using a Gatan Orius CCD camera.

Briefly, the immunogold labelling was performed by placing 4 μL of a dGAE filament preparation for 1 min on Formvar/carbon‐coated 400‐mesh copper TEM support grids (Agar Scientific), and then the excess was removed using filter paper. For blocking, grids were incubated with normal goat serum (1 : 10 dilution) for 30 min at room temperature and then incubated with mAb 423 (1 : 20 dilution) for 2 h at room temperature. (mAb 423 was raised against PHFs from AD brains and detects tau protein C‐terminally truncated at Glu‐391 as reported in 19, 26). Grids were washed three times with PBS+ for 2 min each and then incubated with GaM10 secondary antibody (1 : 10 dilution) for 1 h at room temperature. The grids were washed five times with PBS+ for 2 min each followed by five washes with distilled water (0.22‐μm‐filtered) for 2 min each. The filaments were negatively stained with 2 % (w/v) uranyl acetate (0.22 μm filtered) for 1 min. TEM projection images were collected using a JEOL JEM1400‐Plus Transmission Electron Microscope operated at 100 kV equipped with a Gatan OneView camera (4k × 4k). Images were recorded at 25 fps with drift correction using GMS3.

3.5 μl sample solutions of undecorated or TRIM5-decorated CA tubes were spread onto the carbon side of freshly glow-discharged, Formvar/Carbon-coated, 200-mesh copper grids (Electron Microscopy Sciences). The samples were incubated for 4 min, rinsed briefly by flotation on a drop of 100 mM KCl, blotted dry, stained for 2 min in filtered, saturated uranyl acetate (or 1 min in 1% phosphotungstate), blotted dry, and allowed to air dry. Samples were viewed on a JEOL JEM-1400 Plus transmission electron microscope operated at 120 kV accelerating voltage, and images were acquired as Gatan Digital Micrograph 3 (DM3) files with a Gatan Ultrascan CCD camera or on a Hitachi 7100 TEM at 75 kV accelerating voltage with a Gatan ORIUS CCD camera, and converted into JPEG images using ImageJ software (NIH Bethesda, MD, USA).