Formvar and carbon coated copper grids

Transmission electron microscopy (TEM) sections were prepared as previously described [17 (link)]. Briefly, spheroids were pelleted and vitrified by high-pressure freezing (HPM100, Leica Microsystems) to −90 °C for 80 h in acetone with 1% OsO₄. The temperature was then raised from 1 °C/h to 30 °C and samples were rinsed 4 times in acetone. Samples were infiltrated with agar low viscosity resin (LVR, Agar scientific) in acetone for 3 h. After polymerisation for 24 h at 60 °C, 70–400 nm sections were obtained using an ultra-microtome (UC7, Leica Microsystems). Sections were collected on formvar-carbon-coated 100 mesh copper grids and post-stained for 10 min with 2% aqueous uranyl acetate, rinsed and incubated for 5 min with lead citrate. Grids were analysed using a Tecnai 12 FEI Microscope (120 kV) at different magnifications. 200-mesh formvar and carbon-coated copper grids (Agar Scientific) were incubated for 60 min with a 50 μl drop of the relevant SeNP solution. Grids were washed in ultrapure water for 5 min, three times, and stained with 2% phosphotungstic acid (Agar Scientific) in water, pH 7, for 10 min. Excess solution was absorbed with filter paper and grids were allowed to air-dry overnight. Grids were imaged on a JEM-1400 Flash Transmission Electron Microscope at 120 kV.

Samples of proteins at different time points were placed onto formvar- and carbon-coated copper grids (Agar Scientific Ltd., Stansted, England) and incubated for 5 min. Excess protein was removed using filter paper from the edge of the grid. The grids were washed 3 times with filtered distilled water, and blotted dry on filter paper. Next, negative staining was conducted with 2% filtered uranyl acetate for 2 min. Excess dye was blotted on filter paper, washing was conducted again 3 times, and samples were left to air dry for 10 min. Micrographs were obtained using a 120 kV JEM-1400 transmission electron microscope (JEOL, Tokyo, Japan) at accelerating voltage 80 keV.