Fig. 3. B-Cy3mBSA and F-Dex are internalized into distinct endosomal compartments at very early times. Hemocytes from CS animals were incubated at room temperature with B-Cy3mBSA (A,E; red in D,H) and F-Dex (C,G; green in D,H) for 45 seconds either without (A-D) or with (E-H) a pre-pulse of B-Cy3mBSA for 3 minutes. Cells were then immediately chilled on ice and Cy5-SA (B,F; blue in D,H) was added on ice to detect surface-accessible receptors, prior to fixation and imaging on wide-field microscope. Note that after 45 seconds a significant fraction of the cell-associated B-Cy3mBSA is distributed in endosomes inaccessible to Cy5-SA (A,D and E,H; arrows). Most of the brightest F-Dex-labeled structures (C,D and G,H; arrowheads) do not colocalize with B-Cy3mBSA. A large fraction of the peripherally distributed B-Cy3mBSA is colocalized with Cy5-SA, remaining surface accessible at both times (A,B,D, and E,F,H; small arrowheads). Insets show magnified view of the area marked by an asterisk. Bar, 5 µm; inset, 1 µm.