Protocol for fixation and staining of Drosophila melanogaster embryos optimized for DNA-PAINT nanoscopy of adherens junction proteins

Cadherin-catenin based adherens junctions are dynamic protein complexes that mediate homophilic cell-cell adhesion, strengthen cell-cell contact, and maintain cell polarity. Here, we present a protocol to prepare Drosophila embryo samples for DNA-PAINT nanoscopy to resolve nanoscale E-cadherin clusters at junctions. We describe steps for embryo collection, fixation, immunostaining, and mounting onto glass-bottom chambered slides to prepare samples suitable for DNA-PAINT nanoscopy. DNA-PAINT microscopy provides insight into the nanoscopic clustering and arrangement of E-cadherin complexes in fixed tissue. For complete details on the use and execution of this protocol, please refer to Häring et al. 1