Uranyl acetate

Conjugating pairs of cells expressing GFP–Nup93 were embedded in an agarose gel on a glass-bottomed dish with an addressing grid (grid size, 175 µm) on the coverslip (AGC Techno Glass). Live cells were monitored on the DeltaVision system; to fix cells, 2.5% glutaraldehyde solution was layered over the agarose gel and left for 2 hrs. 3D images (50–60 z-stacks×0.2 µm intervals) were obtained using an oil immersion objective lens (PlanApoN60xOSC/NA1.4; Olympus) and processed by deconvolution. EM observation of the same cells was carried out as described previously (Haraguchi et al., 2008 (link)): Briefly, the cells in the agarose gel were post-fixed with 1% OsO₄ (Nisshin EM, Tokyo, Japan) for 1 hr. After sequential dehydration with ethanol, the samples were stained en bloc with 2% uranyl acetate (Wako, Osaka, Japan) for 30 min. The epoxy block containing the cells of interest was trimmed according to the coordinates of the grid and sliced into 80-nm sections. Image data were collected by JEM-2000EX II (JEOL, Tokyo, Japan) with an acceleration voltage of 80 kV.

Aliquots of 10 µL from the HMW-αSo and LMW-αS fractions were dotted onto a glow-discharged carbon-coated formvar grid (Okenshoji, Tokyo, Japan) and incubated for 20 min. Then, an equal volume of 2.5% (vol/vol) glutaraldehyde solution was added dropwise, and the solution was incubated for 5 min. Finally, the proteins were stained with 8 µL of 1% (v/v) uranyl acetate (FUJIFILM Wako Pure Chemical Corporation). After removing the residual solution and air-drying the grids, the samples were observed under a transmission electron microscope (JEM-1210; JEOL Ltd., Tokyo, Japan).

The EV pellets collected at the bottom of tubes were directly fixed with 100 μL of modified Karnovsky’s fixative solution⁸⁰ (2.0% paraformaldehyde and 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2) for 1 h at 4 °C. After fixation, the pellets were carefully recovered with a spatula and embedded in LR White resin (Agar Scientific, Stansted, Essex, LDN, UK). Ultra-thin sections were prepared with an Ultracut UCT microtome (S9329, Leica, S9329, Wetzlar, Germany) and the specimens were stained with 6% uranyl acetate (Wako, Tokyo, Japan) and 3% lead citrate (Wako) on formvar-coated (Nissin EM, Tokyo, Japan) nickel grids (S-300 square mash, Gilder, Grantham, UK). Transmission electron microscopy (TEM) images were obtained with a H-7650 instrument (Hitachi, Co., Tokyo, Japan).