Internalized granzyme B colocalizes with the endosomal markers, rab4 and rab5 at 20°C. (A) HeLa target cells were incubated for 60 minutes at 20°C to allow uptake of soluble granzyme B-FITC, immunolabeled for rab5 or rab4 with corresponding TR-conjugated secondary antisera and viewed by CLSM. rab5⁺ and rab4⁺ endosomes appear red and sites of granzyme B-FITC appear green. Colocalization of granzyme B and rab5 or rab4 is indicated by the appearance of yellow. Scale bar is 10 μm. (B) Ultrathin sections of Lowicryl embedded HeLa cells treated with granzyme B at 20°C were immunolabeled with polyclonal antisera against rab4 (10 nm gold) and monoclonal antibody against granzyme B (18 nm gold). Endosomal compartments labeled with rab4 (arrowheads) occasionally contain granzyme B (arrows). Some rab4 is seen in the cytoplasm, but the general background is low, as illustrated by the lack of gold particles in the nucleus (N). Scale bar is 1.0 μm.