Vacuum desk 3 sputter coater

A small aliquot (~2–5 μL) of CVM was loaded directly onto poly-L-lysine treated 12 mm diameter German round glass coverslip. The mucus was flattened with a second coverslip and the coverslips separated horizontally. The coverslips were loaded sample side up into a tissue culture plate (24-well). The samples were fixed using 1 mL solution of 2.5% w/v glutaraldehyde, 3 mM MgCl₂, 1% sucrose in 0.1 M Sorensen’s phosphate buffer (pH 7.2). Fixation time was either 1 h at room temperature if post-fixation steps could be performed the same day, or for 24–48 h in the cold room if post-fixation steps were scheduled for a later day, according to scheduling availability with imaging facility staff. After fixative treatment, the solution was removed and rinsed 3 times for 15 min each with a solution of 0.1 M Sorensen’s phosphate buffer (pH 7.2), 3mM MgCl₂ and 3% sucrose. Samples were then postfixed in 1% osmium tetroxide in buffer for 1 h on ice in the dark, rinsed twice with distilled water, and dehydrated with a graded ethanol series. Samples were dried with hexamethyldisilazane (HMDS) and mounted on carbon-coated stubs. Samples were sputter coated with 20 nm AuPd mixture in a Denton Vacuum Desk III Sputter Coater. The samples were imaged in a LEO/Zeiss Field-emission SEM at 1 kV.