DC Endocytosis of SVLP Figure 3. SVLP association with lipid rafts. (A) Pre-chilled DC were treated with 2.5 mg/ml SVLP (green) and 5 mg/ml CTB (red), followed by 30 min incubation on ice and washing. Samples were fixed and analysed by confocal microscopy. Arrows indicate SVLP and CTB localisation. The white square is the zoomed area in the lower panel; the lower panel shows a different section to that in the upper panel, to demonstrate that areas with apparently no co-localisation of the SVLP with CTB could overlay areas showing co-localisation. Scale bars: 50 mm (upper panel) and 5 mm (lower panel). (B) DC treated as (A) were given warm medium and shifted to 39uC for 10 min. Arrowheads indicate SVLP and CTB co-localisation on polarised membranes at intercellular contacts; arrows indicate internalisation at polarisation sites. Scale bars: 10 mm (upper panel) and 5 mm (lower panel). (C) Pre-chilled DC incubated with 2.5 mg/ml SVLP (green) on ice for 20 min followed by washing and shifting to 39uC for 10 min. The image in the lower panel is a zoom of the white square in the upper panel. Arrows show SVLP associated with lamellipodia. Scale bars: 30 mm (left) and 10 mm (right). (D) DC were pulsed with 2.5 mg/ml SVLP and 5 mg/ml CTB for 30 min on ice, then shifted to 39uC for 30 min. High-resolution stacks, 3D images and co- localisation analysis, using IMARIS, demonstrate co-localisation of SVLP with CTB; this included algorithmic analysis to identify co-localised voxels. Scale bar: 8 mm. (E) Algorithmic analysis of the results from (D) performed using IMARIS. doi:10.1371/journal.pone.0043248.g003 more distinctly in the zoomed images of the lower panel for Fig. 3A. This was considered to reflect the irregular distribution of lipid rafts throughout the membrane. These pre-chilled DC were also given SVLP together with CTB for 30 min on ice, washed to remove unbound particles and CTB, and shifted to 39uC for different periods of time. At 0 min (the time of temperature PLOS ONE | www.plosone.org 5 August 2012 | Volume 7 | Issue 8 | e43248