Leica em ac20

Overnight cultures of WT and ΔCbpD in LB were diluted 1:100 and grown in LB to the mid-exponential growth phase. To visualize the effect of serum on bacterial lysis, the bacteria were washed in PBS and suspended in RPMI-HSA supplemented with 10 % (v/v) NHS or HI-NHS for 1 h. The cell pellets were then washed in 0.1 M sodium cacodylate buffer (Sigma) and fixed briefly in a mix of 2% paraformaldehyde (Sigma) and 2.5% glutaraldehyde (Sigma) in 0.1 M sodium cacodylate. Samples were treated with 1% osmium tetroxide (OsO₄; TAAB) before stepwise dehydration in increasing concentrations of ethanol, followed by incubation in acetone. Next, the samples were infiltrated with propylene oxide, embedded using the EMbed 812 resin kit (Electron Microscopy Sciences), and finally polymerized 48 h at 60 °C. Ultrathin sections were collected on copper grids and stained with 2% uranyl acetate and 0.5% lead citrate on an automated contrasting instrument Leica EM AC20 (Leica Microsystems). Finally, the grids were imaged using TEM (Philips CM10 microscope) equipped with a 600-W camera (Gatan) (80–90 kV).

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).

All steps of the immunolabelling were performed in a humid chamber at room temperature. Grids were floated upside down on 25 μl of aliquots of blocking solution (5% BSA, 1% Fish skin gelatin in Phosphate buffered saline (PBS)) for 30 min followed by a wash step for five times 5 min in incubation buffer (IB: 1% BSA in PBS). Incubation of primary antibodies for 120 min, Goat anti-GFP-biotin antibody, 1:300 (Rockland 600–106–215) followed by washing five times 5 min in IB. The grids were then incubated with unconjugated bridging antibodies, Rabbit anti biotin, 1:10,000 (Rockland 100–4,198) for 30 min. After washing five times 5 min in IB, the grids were incubated in Protein A gold (PAG) (10 nm, Cell Biology, Utrecht University) and washed twice, 5 min each, with IB; three times 5 min with PBS; and five times 2 min with double distilled water. Control experiments consisted of treating sections with bridging antibodies and/or PAG 10 nm alone.
After post-staining in a Leica EM AC20 (Leica, Microsystems) for 30 min in uranyl acetate at 20 °C and for 7 min in lead citrate at 20 °C sections were examined with a JEOL JEM 1010 (Jeol, Ltd, Tokyo, Japan) transmission electron microscope operating at 60 kV. Pictures were digitized using a Ditabis system (Pforzheim, Germany).