Localization of VAMP-8 and SG markers in resting and stimulated WT BMMCs. (A) Colocalization of VAMP-8 with the SG markers serotonin and mMCP-6 was analyzed in BMMCs using rat antiserotonin (top panel) and rat antimouse mMCP-6 (bottom panel) using confocal microscopy. Representative single optical sections and overlay (merge) images are shown. (B) Ultrastructural colocalization analysis of RPMCs using VAMP-8 (10 nm GP, black arrows) and mouse antiserotonin (20 nm GP, white arrows). The inset shows VAMP-8 granule matrix labeling on vesicular-like structures. (C) Colocalization of VAMP-8 with SG marker serotonin was analyzed in unstimulated (NS) and PMA/ionomycin (3 minutes)–stimulated (S) BMMCs. Cells were stained with rabbit anti–VAMP-8 and mouse antiserotonin. For comparison, relative localization to plasma membrane marker c-kit (rat mAb) is shown. Cells were analyzed by confocal microscopy. Representative single optical sections as well as 2-color (2C) and 3-color (3C) overlay images are shown. Compartments showing overlap for 2 colors and 3 colors appear as yellow and white, respectively. Bars represent 5 μm.