Phosphotungstic acid

High titer phage lysates (10⁹ – 10¹¹ PFU/ml) were chosen for transmission electron microscopy (TEM) and prepared for imaging as described previously (Deveau et al., 2006 (link)), with modifications. Briefly, one ml of lysate was spun at 4°C for 1.5 h at 21,000 × g. The supernatant was subsequently discarded and the last 100 μl was saved. Consequently, 1 ml of 0.1 M ammonium acetate (Amresco, Solon, OH, United States) was added and the suspension subsequently spun again at 4°C for 1.5 h at 21,000 × g. This purification was repeated twice, with the last 100 μl reserved for TEM.
For grid preparation, 3 μl of purified lysates were placed on carbon coated copper grids (Ted Pella, Redding, CA, United States) following glow-discharge. The phage preparations were subsequently negatively stained with 2% phosphotungstic acid (Ted Pella). A Hitachi H-7600 transmission electron microscope was used for acquiring the images at the University of British Columbia Bioimaging Facility. An accelerating voltage of 80 kV was used for imaging.

EVs were phenotypically and structurally characterized by Transmission Electron Microscopy (TEM) with immuno-gold labelling against CD9, as previously described by Nielsen et al [44 (link)]. Briefly, 5 μl of EV isolate was mounted on a grid (SPI Supplies, PA, USA) and stained with one drop of 1% (w/v) phosphotungstic acid (pH 7.0, Ted Pella, Caspilor AB, Sweden), and subsequently blotted dry on filter paper. To visualize the presence of EV-specific marker CD9 on the surface of vesicles, IEM was performed on the isolated vesicles. The pelleted vesicles were positioned on a grid as described above and then blocked in ovalbumin. Subsequently, the grid was incubated with primary anti-CD9 antibody (1:50, BD Pharmingen, CA, USA), followed by incubation with secondary goat anti-mouse antibody conjugated with 10 nm colloidal gold (1:25, British BioCell, UK). The grids were stained with 1% (w/v) phosphotungstic acid at pH 7.0 and blotted dry. Images were obtained with a transmission electron microscopy (JEM 1010, Germany) operated at 60 keV coupled to an electron-sensitive CCD camera (KeenView, Olympus, PA, USA). Lastly, a grid-size replica (2,160 lines/mm) was imported to ImageJ 1.50i software, which enables a correct determination of the size of EVs.