Protocol for whole-mount staining of melanized insect tissues for RNA and protein detection

Technical barriers have limited whole-mount staining and imaging of internal tissues in many insects and other arthropods. Here, we present a protocol for whole-mount staining of melanized insect tissues for RNA and protein detection. We describe steps for preparing, permeabilizing, and bleaching samples; designing RNA probes; and staining. We then detail procedures for imaging samples. This protocol is compatible with both immunohistochemistry and fluorescence in situ hybridization and enables high-resolution microscopy of tissues surrounded by cuticle without sectioning. For complete details on the use and execution of this protocol, please refer to Glotzer et al. 1 1. Glotzer, G.L. ∙ Pastor, P.D.H. ∙ Kronauer, D.J.C. Transcriptional interference gates monogenic odorant receptor expression in ants Curr. Biol. 2025; 35 :5033-5047.