STORM of SSTR2. (A and D) AtT20 cells were left at steady state (no stimulation) or incubated with 100 nM [D-Trp⁸]-SOM for 60 min. (A) Cells were immunostained for SSTR2 and syntaxin-6 and (B) for SSTR2 and PIST. Cross-nearest-neighbor measurements are shown between SSTR2 and syntaxin-6 versus the spatially unrelated proteins PIST and Giantin (B and C) or for SSTR2 and PIST versus PIST and Giantin (E and F). PM, plasma membrane. Note the increase in spatial association between SSTR2 and syntaxin-6 following [D-Trp⁸]-SOM stimulation in the juxtanuclear clump in B but the random distribution of SSTR2 and PIST in E. Images were acquired using a Vutara SR-350 Biplane point-localization superresolution microscope. Data volumes are displayed in point splatting view. (G) Representative segmentation of juxta-nuclear clump versus the rest of the cells as used for the STORM analysis. Note that the image used to indicate the segmentation in G is identical to the image used to represent the localization SSTR2 and syntaxin-6 in A. (H) The spatially unrelated Giantin and PIST single-molecule localizations are used as a negative control for the distance analysis. Scale bars represent 5 µm.