In situ hybridization methods are used across the biological sciences to map mRNA expression within intact specimens. Multiplexed experiments, in which multiple target mRNAs are mapped in a single sample, are essential for studying regulatory interactions, but remain cumbersome in most model organisms. Programmable in situ amplifiers based on the mechanism of hybridization chain reaction (HCR) overcome this longstanding challenge by operating independently within a sample, enabling multiplexed experiments to be performed with an experimental timeline independent of the number of target mRNAs. To assist biologists working across a broad spectrum of organisms, we demonstrate multiplexed in situ HCR in diverse imaging settings: bacteria, whole-mount nematode larvae, whole-mount fruit fly embryos, whole-mount sea urchin embryos, whole-mount zebrafish larvae, whole-mount chicken embryos, whole-mount mouse embryos and formalin-fixed paraffin-embedded human tissue sections. In addition to straightforward multiplexing, in situ HCR enables deep sample penetration, high contrast and subcellular resolution, providing an incisive tool for the study of interlaced and overlapping expression patterns, with implications for research communities across the biological sciences.
The authors declare competing financial interests in the form of patents and pending patent applications.
Study conceived by N.A.P. in consultation with M.E.B., E.H.D., S.E.F., B.A.H., J.R.L., D.K.N., P.H.P., M.v.d.R., B.W. Experiments designed by H.M.T.C. and N.A.P. Preliminary studies and protocol adaptation performed by S.M.L., R.C.H., A.Z.R., H.M.T.C. (bacteria), C.R.C. (nematode), N.H. (fly), J.C.B., C.R.C. (urchin), H.M.T.C. (zebrafish), T.S.-S., C.R.C. (chicken), A.C.T.A., B.E.D., D.H., H.M.T.C. (mouse), and H.M.T.C., N.H. (human), in consultation with D.K.N., J.R.L. (bacteria), M.K. (nematode), R.L., S.E.F. (chicken), R.L., C.R.C., S.E.F., B.W. (mouse), M.v.d.R. (human), and H.M.T.C., N.A.P. (all organisms). Final protocols optimized and final data collected by H.M.T.C. (bacteria, zebrafish, mouse), C.R.C. (nematode, urchin, chicken) and N.H. (fly, human). Final data analyzed by: S.M.L., S.K.M., R.C.H., D.K.N., A.Z.R., J.R.L. (bacteria), M.K., P.S., C.R.C. (nematode), O.S.A., B.A.H., N.H. (fly), J.C.B., C.R.C. (urchin), D.A.P., S.E.F. (fish), T.S.-S., M.E.B., C.R.C. (chicken), A.C.T.A., B.W., B.E.D., D.H., Y.L., C.R., R.L., S.E.F. (mouse), A.C.M., M.v.d.R., N.H. (human) and H.M.T.C and N.A.P. (all organisms). Paper and supplementary information written by H.M.T.C. and N.A.P. Paper was edited and approved by all coauthors.
This work was funded by the National Institutes of Health (NIH) [5R01EB006192]; the National Science Foundation Molecular Programming Project [NSF-CCF-1317694]; the Gordon and Betty Moore Foundation [GBMF2809]; the Beckman Institute at Caltech (PMTC); the Translational Biomedical Imaging Laboratory at CHLA; the Translational Imaging Center at USC; a Christensen Fellowship at St Catherine's College, University of Oxford; and by the John Simon Guggenheim Memorial Foundation. Deposited in PMC for release after 12 months.
Supplementary information available online at http://dev.biologists.org/lookup/doi/10.1242/dev.140137.supplemental
- Received May 20, 2016.
- Accepted August 1, 2016.