Double In Situ Detection of Sonic Hedgehog mRNA and pMAPK Protein in Examining the Cell Proliferation Signaling Pathway in Mouse Embryo

Double in situ detection of RNA molecules and proteins in tissue sections is not trivial. A successful experiment heavily depends on the preparation of the tissue as well as the quality of the probes and antibodies. Detection of two or more molecular markers also requires reagents and experimental conditions that will preserve authenticity (accuracy) of the single staining patterns. Here, we describe in detail the protocols used to detect sonic hedgehog (Shh ) mRNA by in situ hybridization and immunofluorescence staining for phosphorylated mitogen-activated protein kinase (pMAPK) in the same mouse embryonic tissue sections. In addition to protocols for manual immuno-staining, we provide data from automated machine-based staining protocols and highly recommend it to achieve strong signal and reproducible results.