Glyoxal-based fixation of Drosophila embryos for immunofluorescence staining and RNA in situ hybridization

The dialdehyde glyoxal is an alternative chemical fixative that cross-links tissues faster than formaldehyde, retains higher antigenicity, and is less hazardous than either formaldehyde or glutaraldehyde. Here we present a glyoxal-based fixation protocol for use with Drosophila embryos. We describe steps to prepare acid-free glyoxal, fix embryos, and then stain with antibodies for immunofluorescence (IF). We also describe methods for RNA fluorescence in situ hybridization (FISH) and FISH plus IF (FISH-IF) using glyoxal-fixed embryos. This protocol was adapted for Drosophila embryos from the methods of Bussolati et al.¹ and Richter et al.².