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. 2021 Jun 25;12:3970. doi: 10.1038/s41467-021-24167-9

Fig. 3. Nanogold-based platinum replica protein labeling at the plasma membrane.

Fig. 3

a A schematic of plasmids used for the protein of interests (POI) with histidine and GFP fused at the N or C terminal domains. b Scheme showing dense-core vesicle-associated protein labeled with Ni-NTA-Au. c His-GFP-Rab27a, mCherry-Rab27a, and overlay images, showing colocalization with DCVs. d Colocalization of His-GFP-Rab27a and Anti-Histidine-DyLight 488 showing the accessibility of histidine epitope. The validation experiments were done once. Scale bars are 3 μm. Enlarged small section (white box) for each image is at the top right corner. Scale bar is 0.5 μm. Platinum replica images of HeLa cells (crop from larger PREM image of a cell in Supplementary Fig. 12) expressed with e His-GFP-Clathrin Light Chain A, f His-Cavin-GFP, and g EPS15-GFP-His. Left panel scale bar is 200 nm. Enlarged images in the middle panel show gold particles on endocytic structures marked with orange circles. The same enlarged images in the right panel show the structures without orange markings. Scale bar is 150 nm. Tomogram section (XY view, scale bar = 100 nm) of an individual clathrin structure labeled with Ni-NTA-Au for h clathrin light chain A, i cavin1, and j EPS15, and the orthogonal views in XZ (scale bar = 100 nm) and YZ (scale bar = 50 nm) dimensions. Cyan dashed lines mark the gold particles seen in XY view of a z slice and denote their location in orthogonal views. Two independent imaging experiments were performed for 2D- and 3D-EM.