Propylene oxide

Juveniles processed for TEM were fixed in 4% glutaraldehyde as described above, and then washed in sodium cacodylate buffer at 4°C (60 min). At this point juveniles were cut either longitudinally or transversely before fixation was continued for a further 11 h at 4°C. Juveniles were then washed in 0.1 M sodium cacodylate buffer containing 3% sucrose overnight (~16 h, 4°C) and then stained in 1% OsO₄ and processed through an ethanol series as described above. At this point juveniles were given two washes in propylene oxide (Agar Scientific) for 5 min at 4°C before embedding in resin (25.2% MNA, 25.2% DDSA, 49.6% agar resin and 1% DMP; Agar Scientific) and propylene oxide in a 1:1 ratio. This was left overnight (~16 h) at RT to allow excess propylene oxide to evaporate. Fresh resin was then placed on the samples and they were left for a further 24 h at RT. Juveniles embedded in resin were polymerised at 60°C for 48 h. Ultrathin sections, 60–70 nm in thickness, were cut on a Reichert Ultracut E ultramicrotome, mounted on bare 200-mesh copper grids, double-stained with alcoholic uranyl acetate (5 min) and aqueous lead citrate (3 min) and viewed in a FEI CM 100 TEM operating at an accelerating voltage of 100 kV.

After 18 days of storage in 4% paraformaldehyde at 4°C, biopsy tissues were cut into
0.5-mm³ pieces under EM fixative (2% glutaraldehyde, Agar Scientific, in
phosphate buffer) using a dissecting microscope in a fume hood. Tissue pieces were left at
room temperature overnight in glutaraldehyde before further processing. The next day,
following a 90-minute osmium tetroxide wash (Agar Scientific), samples were thoroughly
dehydrated in a graded series of ethanols (70% 45 minutes, 90% 15 minutes, 3 × 100% 15
minutes each) at room temperature and the ethanol then replaced with propylene oxide (Agar
Scientific). Infiltration started with 60-minute wash in 50% propylene oxide/50% fresh
Agar 100 epoxy resin (Agar Scientific) at room temperature with agitation. After a further
60 minutes in pure resin, samples were placed into Beem capsules (Agar Scientific) and
polymerized at 60°C overnight. Thin sections (70 nm) were cut using a Leica UC6
ultramicrotome, stained with uranyl acetate and lead citrate in a Leica EM Stain, and
imaged at 100 kV in a FEI T12 TEM with Tietz 2 k × 2 k CCD camera.

KC cells at a density of 7 × 10⁵ cells were seeded on 13 mm Thermanox plastic coverslips (Thermo Fisher Scientific, Swindon, UK) and incubated overnight at +28 °C. Cells were then infected at a MOI of 5 with BTV-1, BTV-26 or rBTV-1_{26 S2,S6,S7} then incubated at +28 °C. At two days pi cells were fixed in phosphate buffered 2% glutaraldehyde (Agar Scientific Ltd., Stansted, UK) for 1 h followed by 1 h in aqueous 1% osmium tetroxide (Agar Scientific Ltd., Stansted, UK). The samples were dehydrated in an ethanol series; 70% for 30 min, 90% for 15 min and 100% three times for 10 min each. A transitional step of 10 min in propylene oxide (Agar Scientific Ltd., Stansted, UK) was undertaken before the samples were infiltrated with a 50:50 mix of propylene oxide and epoxy resin (Agar Scientific Ltd., Stansted, UK) for 1 h. After a final infiltration of 100% epoxy resin for 1 h, the samples were embedded and polymerised overnight at 60 °C. Eighty µm thin sections were cut, collected onto copper grids (Agar Scientific Ltd., Stansted, UK) and grid stained using Leica EM AC20 (Leica Microsystems, Wetzlar, Germany) before being imaged at 100 kV in a FEI Tecnai 12 TEM with a TVIPS F214 digital camera (FEI Company, Hillsboro, OR, USA).