Immunoelectron microscopy of granzyme B in its intracellular compartment. (A) HeLa cells were treated with granzyme B as in Fig 4B, except that after binding of granzyme B to the cell surface at 4°C, cells were moved to 37°C to allow transport to its retention compartment. Labeling with antibody against granzyme B followed by 10 nm gold-conjugated secondary antibody demonstrates that granzyme B is retained in a compartment with morphologic characteristics different from rab4⁺ endosomes shown in Fig 4B. (B) HeLa cells were treated with granzyme B as in (A). Sections were incubated with antibodies to granzyme B and antisera against rab5 followed by corresponding secondary antibodies conjugated to 10 nm and 18 nm gold particles for granyzme B and rab5, respectively. At 37°C, granzyme B, denoted by arrows, was found primarily in a compartment not containing rab5. However, the more electron dense endosomal vesicles containing rab5 (denoted by filled arrows) are only occasionally labeled with granzyme B (open arrowhead). The morphology of the compartment containing granzyme B is distinct from the rab5 compartment. As a negative control in both panels, sections were labeled with each gold-conjugated secondary antibody in the absence of the corresponding primary antibody or antiserum. Sections treated in this manner did not display any labeling (data not shown). Scale bars are 0.1 μm.