DC Endocytosis of SVLP Figure 5. SVLP associating with early endosomes, MHCII and DQ-Ova. (A) Application of quenched SVLP signal to identify endosomal enzyme activity leading to de-quenching of the signal. DC were incubated with 2.5 mg/ml quenched SVLP (green) for different incubation times at 39uC. Samples were fixed, permeabilised and labelled with antibody targeting EEA-1 (red). Acquisition of the images was by confocal microscopy, followed by analysis and merging using IMARIS. Scale bars: 30 mm. (B) Cells were treated as for (A), but also with DQ- Ovalbumin. Acquisition of the images was by confocal microscopy, followed by analysis and merging using IMARIS. Scale bar: 20 mm. (C) Algorithmic co-localisation analysis of SVLP and DQ-Ova performed using IMARIS. (D) Cells were treated as for (A), but stained also for MHCII molecules (blue). Acquisition of the images was by confocal microscopy; high-resolution stacks were prepared using IMARIS. Scale bars: 20 mm (top left), 5 mm (zooms). doi:10.1371/journal.pone.0043248.g005 This enzymatic degradation of SVLP in vesicular structures (Fig. 5A) was relatively slow compared with the rapid internalisation (see Fig. 2D). Accordingly, the SVLP accumulation was further analysed together with endocytosed DQ-Ova, another fluoro- chrome-quenched molecule used to identify the action of endosomal enzymes. Considering the inefficient binding of ova to DC on ice, only the SVLP were pulsed with DC on ice, followed by washing and addition of the DQ-Ova in the medium at all times of incubation. After 30 min at 39uC, many of the SVLP-positive vesicular structures also contained de-quenched (enzymatically cleaved) DQ-Ova (Fig. 5B). The association was particularly strong at 60 min, and continued to be observable at 8 hrs of incubation (Fig. 5B). Application of co-localisation algorithm confirmed this close association (Fig. 5C), giving a Pearson’s coefficient in co- PLOS ONE | www.plosone.org 8 August 2012 | Volume 7 | Issue 8 | e43248