FIGURE 3.

Different colocalization of CD9 with NTAL or LAT. BMMCs were prefixed in 2% paraformaldehyde and then stained with 2H9 mAb followed by secondary antibody conjugated to 12 nm of gold (A, D, G, and J). Alternatively, the cells were first treated with 2H9 mAb and then fixed and stained (B, E, H, and K) or the cells were first treated with 2H9 mAb followed by its aggregation with secondary antibody followed by fixation (C, F, I, and L). Plasma membrane sheets were then isolated and NTAL (A-F) or LAT (G-L) on the cytoplasmic side of the plasma membrane were labeled with primary antibodies followed by secondary antibodies conjugated to 6-nm gold particles. Topography of gold particles was evaluated by electron microscopy. Representatives from 3 independent experiments performed are shown (A-C and G-I). Evaluation of colocalization of 6- and 12-nm gold particles is represented as PCCF analysis for CD9/NTAL labeling (D-F) and CD9/LAT labeling (J-L). For calculation of PCCF, 20 μm² of the plasma membrane sheets was used in each experiment. PCCF indicates colocalization when experimental values (solid line) are higher than random distribution of particles presented by a dotted line. Bars, 200 nm.