PREM images supporting data in figure 6 of "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells"

This dataset is part of a collection of imaging data (https://doi.org/10.25444/nhlbi.c.5405490) from the publication entitled: "The nanoscale molecular morphology of docked exocytic dense-core vesicles in neuroendocrine cells." by Prasai et al. 2021 (https://doi.org/10.1038/s41467-021-24167-9). It contains EM images for PC12 cells expressing His-GFP-Syntaxin1A in fig 6a, and for His-GFP-SNAP25 in fig. 6d. These proteins expressing cells were treated with Ni-NTA-Au for gold labeling.HeLa cells were transiently transfected with His-tagged Syntaxin1A and SNAP25. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. Cells were blocked in 3% BSA/PBS solution for an hour, incubated in a sonicated (5 min) 1:5 solution of 10 nm Ni-NTA-Nanogold in PBS for 1 h. TIRF map was made and samples placed in 2% glutaraldehyde. Samples were prepared for EM images next that included staining with tannic acid and uranyl acetate, dehydration with ethanol and critical point drying. Dried samples were coated with platinum/carbon, replica lifted, transferred to EM grid and 2D TEM images taken at 15,000× magnification (1.2 nm per pixel) using a JEOL 1400 and SerialEM freeware for montaging. 2D TEM was used to survey the gold tagged organelles and tomograms were collected for those cells. Single-axis tilt series (−60° to 60°, 1° increments) were collected at 8,000×. The montages were stitched together, and the tilt series were reconstructed into tomograms using IMOD software.