In situ hybridization (ISH) techniques allow for the study of the nucleic acid expression within whole biological samples. The quality of probes for ISH dictates how accurate and bright the signal is for the experiment; however, there is currently not a systematic way to determine what the best probe set would be. In response to this, we have developed a framework to optimize an ISH probe set to achieve the greatest signal-to-background ratio. As methods like ISH help obtain more information about biological processes, there is a growing desire to simultaneously analyze various targets within the same sample to examine these complex genetic interactions. To facilitate this, a novel amplification technique called hybridization chain reaction (HCR) has allowed for the in situ detection of multiple target mRNAs concurrently in zebrafish embryos. We have now expanded this technology further by adapting HCR amplification for ISH to other model organisms, particularly, whole mount Drosophila melanogaster embryos and formalin-fixed parafin-embedded human tissue sections. Beyond looking at mRNA, immunohistochemistry (IHC) provides another tool to understand biological systems by analyzing protein expression patterns. The ability to easily look at both mRNAs and proteins in the same sample offers significant advantages as each provides unique information, but current methods are technically difficult and labor intensive. In response, we have engineered a scheme to use HCR to amplify signal for IHC. We then used this advancement to develop a straightforward protocol using HCR amplification for simultaneous detection of multiple proteins and mRNAs with a high signal-to-background ratio
