Protocol for dissecting Drosophila pupae and visualizing RNA expression using hybridization chain reaction

Visualizing RNA expression in the Drosophila epidermis during pupal development is challenging because the tissue is fragile during early pupal stages and increasingly impermeable at later stages. Here, we present a protocol for tissue dissection and detection of RNA in situ. We describe steps for using the hybridization chain reaction (HCR) in early and late Drosophila pupal stages (and larval imaginal discs). This protocol facilitates the study of dynamically changing patterns in gene expression during pupal development.