TIRF and PREM images supporting data in figure 3 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) <br>It contains raw TIRF data for His-tag validation in fig. 3c,d. It also contains PREM images for fig 3e-g that show HeLa cells expressing Clathrin light chain A, Cavin1, and EPS15, respectively that were tagged with Ni-NTA-Au.<br>For TIRF correlation data, PC12 cells were were maintained in growth media containing DMEM, 10% fetal bovine serum, and 1% vol/vol penicillin/streptomycin. They were transiently transfected with His-GFP-Rab27a and mCherry-Rab27a to check for colocalization with DCVs. Cells were rinsed in intracellular buffer and manually unroofed with 19-gauge needle and syringe using 2% paraformaldehyde. PC12 cells transfected with His-GFP-Rab27a were treated with Anti-Histidine-DyLight488 to test for accessibility of histidine epitope. TIRF imaging performed using an Olympus IX-81 inverted fluorescence microscope.<br>For PREM data, HeLa cells were transiently transfected with His-GFP-Clathrin Light Chain A, His-Cavin-GFP, and EPS15-GFP-His. 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, 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.