Duplex in situ hybridization in the study of gene co-regulation in the vertebrate brain.

We describe here a high-sensitivity in situ hybridization protocol, optimized for fresh-frozen brain sections, that enables the detection of two transcripts, at single cell resolution. Riboprobes directed against two mRNAs of interest are synthesized with nucleotides tagged with different haptens (digoxigenin- or biotin-UTP), via in vitro transcription, hybridized simultaneously to brain sections, and independently detected through immunocytochemistry. Sequential detection of each probe involves peroxidase-mediated precipitation of tyramide-linked fluorophores of separate emission wavelengths. In addition, we demonstrate how classic non-fluorescent chromogens, such as 3,3'-diaminobenzidine, can be successfully combined with fluorescence-based detection, to yield reliable detection of two transcript populations. We provide examples of representative results obtained with this protocol and describe necessary controls. Additionally, we discuss common problems associated with this methodology, and detail troubleshooting recommendations. Although this method has been optimized for brain sections, it may be useful to detect two mRNA species in a variety of tissues.