Immunocytochemistry of N-terminally HA-tagged glycoprotein-encoding Oropouche virus medium (M) segment co-transfected into HeLa cells with C-terminally YFP-tagged charged multivesicular body (CHMP) proteins, or co-stained with endogenous CHMP6. For all images, CHMP proteins are visible in the 488 nm channel and HA-tagged OROV glycoproteins are visible in the 568 nm channel. GM130, a marker of the Golgi apparatus ('Figure 5'), or TGN46, a marker of the trans-Golgi network ('Figure 6'), are visible in the 647 nm channel.

HeLa cells were seeded on glass coverslips at a density of 5×10^4 cells per well in 24 well dishes. Cells were transfected with 250 ng of DNA (split evenly by mass between the plasmids indicated) using TransIT-LT1 (Mirus) and incubated for 24 h. Where indicated, cells were treated for 6 h prior to fixation with 1 uM monensin to disrupt protein transport from the Golgi. Cells were transferred onto ice and were washed with ice-cold PBS and fixed with cold 250 mM HEPES pH 7.5, 4% (v/v) electron microscopy-grade formaldehyde (PFA, Polysciences) for 10 min on ice before and then incubated with 20 mM HEPES pH 7.5, 4% (v/v) PFA at room temperature for a further 20 min. After washing with PBS, cells were permeabilized by incubation with 0.1% saponin in PBS for 30 min before being incubated with blocking buffer (5% [v/v] FBS, 0.1% saponin in PBS) for 30 min. Primary antibodies were diluted in blocking buffer and incubated with coverslips for 2 h. Coverslips were washed five times with blocking buffer before incubation for 1 h with the relevant secondary antibodies diluted in blocking buffer. Coverslips were washed five times with blocking buffer, three times with 0.1% saponin in PBS, three times with PBS, and finally with ultrapure water. Coverslips were mounted using Mowiol 4-88 (Merck) containing 200 nM 4′,6-diamidino-2-phenylindole (DAPI) and allowed to set overnight. Cells were analysed on a Zeiss confocal laser scanning microscope (LSM)780 (Zeiss).