Fig. 1. ANF-EGFP marks a subgroup of DCVs in cultured hippocampal neurons. (A) Single ANF-EGFP-labeled DCVs visualized by TIRF microscopy. Left panel: vesicular localization of ANF-EGFP in both the soma and neurites of hippocampal neurons. Right panel: fluorescent beads 100 nm and 400 nm in diameter visualized under the same TIRF settings. (B) Immunocytochemical characterization of ANF-EGFP puncta. Row 1: all ANF-EGFP-containing vesicles were positive for the DCV marker SgII. Row 2: all ANF-EGFP-containing vesicles contained the Ca²⁺ sensor SytI. Row 3: ANF-EGFP-containing vesicles were distinct from monoaminergic VMAT2-positive vesicles. Row 4: most ANF-EGFP-containing vesicles also contained immunoreactive BDNF. Fluorescence line scans are shown in insets. These indicate that all ANF-EGFP-containing vesicles (green) also contained SgII and SytI (red). All images were visualized by TIRF microscopy. Scale bars: 1 µm.