Protocol for multiplex whole-mount RNA fluorescence in situ hybridization combined with immunohistochemistry in the mosquito brain

Advances in sequencing technologies have enabled transcriptional profiling of previously understudied yet critical species, such as mosquitoes. Here, we present a protocol for multiplex whole-mount RNA fluorescence in situ hybridization combined with immunohistochemistry in the Anopheles gambiae brain. We describe steps for dissection, fixation, probe hybridization, hybridization chain reaction (HCR) probe detection, and primary antibody application. We then detail procedures for tissue preparation and imaging. The protocol is optimized for assessing 3D spatial gene expression in mosquito brains.