Ultrathin frozen sections of human platelets immunolabeled with monoclonal antibodies to clathrin followed by polyclonal antibodies to Lck (a and b) or with the reverse sequence of label (c). Sections were incubated with either monoclonal antibodies to clathrin (a and b) or polyclonal antibodies to Lck (c) followed by appropriate bridge antibody and 5-nm immunogold particles. After blocking in 1% glutaraldehyde in PBS for 10 minutes, the sections were immunolabeled with either polyclonal antibodies to Lck (a and b) or monoclonal antibodies to clathrin (c), followed by appropriate bridge antibody and 10-nm immunogold particles. In all figures, 5-nm gold particles are indicated by small arrows and 10-nm gold particles by large arrows. (a) to (c), Label for clathrin and Lck are associated. (c) Label for Lck is in clusters (small arrows) and clathrin is associated with these clusters (large arrows). Similar results were obtained with protein A–gold or IgG-gold probes. In no case was the level of 10-nm gold the same as in a single label for the same primary antibody, probably due to steric hindrance of the gold particles. G, granule. (a) Original magnification × 84,000; (b) × 84,000; (c) × 117,000.