Figure 3.

HA11 antibody uptake endocytosis assay in dopaminergic neurons. Mes-Str cultures were transfected with YFP-HA-DAT and assayed after 3 d (see Materials and Methods). The cells were incubated with HA11 for 60 min at 20°C, washed, further incubated for 30 min at 37°C, and fixed. The nonpermeabilized cells were stained with Cy5-tagged anti-mouse secondary IgG. After Triton X-100 permeabilization, the cells were incubated with the same secondary IgG tagged with Cy3 and simultaneously incubated with rabbit antibody to TH. Finally, the cells were incubated with secondary anti-rabbit IgG conjugated with Alexa-350. A z-stack of 30 x-y images was acquired through four filter channels. The main image (scale bar, 10 μm) and high-magnification (scale bar, 3 μm) images of cell soma (left of the main image) represent optical section 10. An inset at the right bottom corner of the main image represents an image of the TH staining. High-magnification images of a part of the processes (right of the main image) represent optical section 7 (scale bar, 3 μm). Green, cyan, and yellow arrows point to examples of compartments that contain, respectively, only YFP (total DAT), YFP colocalized with Cy5 (surface YFP-HA-DAT), and YFP colocalized with Cy3 (internalized HA11). A white arrow shows an example of an axonal varicosity with overlapping YFP, Cy5, and Cy3 fluorescence.