Protocol for multiplex whole-mount RNA fluorescence in situ hybridization combined with immunohistochemistry in the mosquito brain
Anya Suppermpool, Chintan A. Trivedi, Gareth T. Powell, Marta Andrés
Abstract
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. • Step-by-step workflow for combined in situ HCR and immunohistochemistry • Simultaneous visualization of mRNA and protein in whole brain tissue for An. gambiae • Designer tool for customizable and cost-effective HCR probes Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.