Localization of internalized anti-GFP nanobodies in VAMP2–pHluorin–expressing hippocampal neurons. (A) Hippocampal neurons expressing VAMP2–pHluorin were subjected to sdTIM, fixed, and immunolabeled against endogenous Synapsin-1 (pseudocolored in blue). The merged image shows an overlay of the images. Bar, 5 µm. The boxed areas are shown with higher magnification in B and C. Bars, 1 µm. (B) VAMP2–pHluorin (i) colocalized with Synapsin-1 (ii) in active presynapses containing VAMP2–pHluorin–bound Atto647N nanobodies (iii), as seen in the merged image (iv). (C) Presynapses positive for Synapsin-1 (ii), but not for VAMP2–pHluorin (i), did not contain Atto647N nanobodies (iii), as shown in the merged image (iv). Hippocampal neurons were grown on gridded glass-bottom dishes and transfected with VAMP2–pHluorin. (D) The localization of live VAMP2–pHluorin–positive neurons on the grids was recorded, and the cells were then subjected to sdTIM using HRP-mCherry-GNT. After fixation and cytochemical staining, the neurons were processed for EM. VAMP2–pHluorin expression is shown at higher magnification in the inset from the indicated boxed area. Bar, 10 µm. (E) The same neuron was then located in the electron micrograph by following the recorded location of the cells on the grid. (F) HRP precipitate in SVs (arrows), residual staining (open arrowheads) on the plasma membrane (PM), unstained vesicles (arrowheads), and bulk endosomes (e) are indicated. The presynapse shown in E and F is indicated with the tilted box in D. Bars, 0.5 µm. LM-EM, light microscopy and EM.