Multiplexed in situ hybridization using HCR

In situ hybridization methods enable the mapping of mRNA expression within intact biological samples. Multiplexed in situ amplification based on the mechanism of hybridization chain reaction (HCR) enables simultaneous mapping of up to five target mRNAs in diverse organisms and imaging contexts, including whole-mount vertebrate embryos.

Simultaneous mapping of four target mRNAs within a fixed whole-mount zebrafish embryo. (movie)
Multiplexed mRNA expression maps using in situ HCR. (fly movie) (urchin movie) (zebrafish movie) (chicken movie) (mouse movie)
How HCR in situ amplification works

DNA probes complementary to mRNA targets trigger chain reactions in which fluorophore-labeled DNA hairpins self-assemble into tethered fluorescent amplification polymers. The programmability and sequence specificity of these amplification cascades enable multiple HCR amplifiers to operate orthogonally at the same time in the same sample.

HCR properties in whole-mount zebrafish embryos
  • robust multiplexing for five target mRNAs at a time
  • deep sample penetration
  • high signal-to-background
  • sharp signal localization