Formvar carbon coated 400 mesh copper grids

Electron microscopy grids were prepared by placing 4 μl of the sample onto formvar/carbon-coated 400-mesh copper grids (Agar Scientific Ltd, UK), followed by two washes with 0.22-μm-filtered milli-Q water. Then, 4 μl of uranyl acetate (2% w/v) was added to the grid and left for 30 s before blotting. EM projection images were collected using a JEOL JEM1400-Plus Electron Microscope operated at 80 kV equipped with a Gatan OneView camera (4k × 4k). Images were recorded at 25 fps with drift correction using GMS3.

EV samples were prepared for transmission electron microscopy on formvar carbon-coated 400-mesh copper grids (Agar Scientific, Stansted, United Kingdom) which were treated with plasma to render the carbon surface hydrophilic. EVs (3 μL) were placed onto a carbon grid and incubated for 20 min for adsorption, with the grids rinsed 3 times in Sorenson’s buffer and fixed with 1% (w/v) glutaraldehyde for 5 min. Again, the grids were washed; 8 times in ultrapure water. The samples were stained using 2% (w/v) uranyl acetate (UA) for 5 min after which 0.5 µL final embedding solution (ES; 700 µL of 2% (w/v) methyl cellulose, 100 µL 3% UA, 187 µL Milli-Q water, 12.5 µL of 2% Phosphotungstic acid) was applied. Excess ES was removed, and the grids were air-dried for 15 min. The samples were analysed using a Tecnai 12 Transmission Electron Microscope, operating at an accelerating voltage of 120 kV. Images were acquired using a Megaview III CCD camera and AnalySIS camera control software (Olympus).

Formic acid extracted hemolymph samples were prepared for TEM negative stain to quantify soluble, oligomeric Aβ within the animals’ body fluids. 4 μL of each sample were pipetted on to Formvar/carbon coated 400-mesh copper grids (Agar Scientific, Essex, UK) for 1 minute. Excess liquid was removed with Whatman paper. Grids were washed with 4 μL of Milli-Q water and blotted, followed by 4 μL of filtered 2% (w/v) uranyl acetate for 1 minute and blotted again. Grids were allowed to air dry before being examined in a Hitachi 7100 TEM at 100 kV and digital images acquired with an axially mounted (2K x 2K pixel) Gatan Ultrascan 1000 CCD camera (Gatan UK, Oxford, UK). After initial imaging, the samples were immunogold labeled (as previously described) to determine oligomeric structure. A 1 μg/mL mouse Nu1 primary antibody (Klein laboratory)16 (link) and a goat anti-mouse 10 nm gold-conjugated secondary antibody (BBI Solutions OEM Ltd., Cardiff, UK) were used and grids were imaged as stated previously.
Negative staining of Aβ 1-42 and Aβ 25-35 was used to determine peptide morphology. Aliquots of 94 μM Aβ 25-35 and 100 μM Aβ 1-42 were allowed to incubate in normal saline solution for 0, 3, or 24 hours. Samples were prepared and images acquired as stated above.

Four μl aliquots of α-syn samples were placed onto Formvar/carbon coated 400-mesh copper grids (Agar Scientific, Essex, UK) for 1 min, and the excess was removed using filter paper. Subsequently the grid was washed using 4 μl of Milli-Q water filtered with 0.22 μM filter and blotted dry, then negatively stained twice with 4 μl of filtered 2% (w/v) uranyl acetate for 1 min and blotted dry. The grid was allowed to air-dry before examination on a Hitachi 7100 transmission electron microscope (Hitachi, Germany) fitted with a Gatan Ultrascan 1000 CCD camera (Gatan, Abingdon, UK) at an operating voltage of 100 kV.