Figure 1: Overview of expansion microscopy in Drosophila embryos.

ExM is a multi-step protocol that takes at least 4 days to complete. Embryo collection, fixation, and devitellinization take 1 day or more depending on whether embryos from multiple collections are pooled. Immunofluorescence labeling takes 1 day or 2 days depending on whether the embryos are incubated overnight with the primary antibodies. Embryo activation, gelation, digestion, and expansion can be performed in a single day. The gels can be mounted and imaged immediately after expansion, although for practical reasons it is often desirable to start imaging the following day.