Some AmpA protein is found extracellularly on the cell surface. A)Live cells were incubated with the membrane stain DiI and with ratanti-RFP antibody in the dark at room temperature. Cells were washed andthen incubated for 1 hour with goat anti-rat antibody conjugated toAlexa-Fluor 488, then washed and fixed but never permeabilized. Imagesrepresent single optical sections of a z-series. Arrows indicatecolocalization of mRFP-AmpA with DiI at the cell periphery. Scale bar is13 um in the field images (top row) and 5 um in the zoom of asingle cell (bottom row). B and C) AmpA on the cellsurface is endocytosed and traffics to a perinuclear endosomal recyclingcompartment. Live cells were incubated 4°C with rat anti-RFPantibody. B) Some of the coverslips were maintained at 4°Cto prevent endocytosis, washed to remove excess anti-mRFP antibody,fixed and then permeabilized and labeled with goat anti-rabbit secondantibody. Slices of cells from a Z series to show that the labeling islargely on the outside of the cell and not in the interior. Scale bar is10 um C) Cells on the other coverslips (panels labeled 22degrees) were washed to remove excess anti-mRFP antibody, then incubatedat room temperature for 15 min to allow endocytosis of the antibodybound mRFP-AmpA and then fixed and permeabilized to allow labeling withsecond antibody to detect anti-RFP antibody that had been internalizedwith the AmpA. The image is a slice from a Z series. The scale bar is10 um.