Fig. 4.

Intracellular localization of HER2-Fc in iDCs after FcγR-mediated internalization. Immunofluorescence analysis of HER2-Fc intracellular localization in DCs after 2 or 12 h from the uptake. The HER2-Fc protein is visualized in green, while the HLAI compartment (identified by calreticulin and HLAI staining) or the endolysosomal HLAII compartment (LAMP1 staining) are immunolabeled in red. 3D reconstruction of a selection of three central sections crossing the nucleus out of the total number of the serial optical sections is shown in each image. A quantitative analysis of the percentage of colocalization was performed by serial optical sectioning and 3D reconstruction. Results are expressed as mean values ± SE (standard errors); the percentage of colocalization was calculated analyzing a minimum of 30 cells for each treatment randomly taken from three independent experiments. After 2 h of internalization, the HER2-Fc staining appears in dots clustered in the perinuclear area of the cells. HER2-positive spots colocalize with the HLAI compartment (57% colocalization with HLAI and 29% colocalization with calreticulin) and only partially with the HLAII compartment (17% colocalization with LAMP1). Arrows point to yellow double-positive spots. After 12 h of internalization, HER2-positive dots are more scattered throughout the entire cytoplasm and the colocalization with HLAI compartment appears only slightly increased. Bar, 10 μm