Sis megaview 2 digital camera

5×10⁶ DCs were challenged with PRU-RFP or RH-DDYFP-Tg14-3-3 (+Shield1) for 24h (MOI 1). Cells were fixed in 4% PFA, 0.1% glutaraldehyde (GA) in 0.1 M phosphate buffer for 60 min. Cells were washed, collected and cryosectioned as previously described (Goldenberg et al., 2007 (link)). Sections were cut at 90 nm thickness and picked up in a 1:1 mixture of 2% methylcellulose and 2,5M sucrose, placed on formvar-coated nickel grids (3mm diameter) and stored at 4°C. For immunolabelling, grids were washed as previously described (Goldenberg et al., 2007 (link)) and blocked in blocking buffer containing 5% skim milk powder, 1% BSA-c (Aurion). Rabbit anti-GFP (Abcam), was added to the grid at 2.5 μg/ml in 5% skim milk powder, 0.1% BSA-c (dilution buffer) for 1.5 h. Following PBS washes, the samples were blocked again. The secondary antibody conjugated to 12 nm colloidal gold (Jackson ImmunoResearch) was diluted 1 in 20 and incubated with the samples for 1 hour. After PBS washes, the cells were fixed with 2% GA, washed with PBS, dH₂O, and 1.8% methylcellulose, 0.2% uranyl acetate in dH₂O on ice. Grids were examined using a Hitachi 7500 transmission electron microscope operating at an accelerating voltage of 80 kV. Images were obtained using an Olympus SIS Megaview II digital camera and iTEM software