Fig. 2.

RNA detection at depth by hybridization chain reaction. a The probe hybridization step and the HCR amplification step. In the hybridization step, ~ 10–20 DNA probes bind to the target RNA. These probes carry the initiators for the HCR amplification step. In the HCR amplification step, each initiator triggers the binding of two hairpin types in order to amplify the fluorescence signal. b Detection of SCG10 mRNA transcripts in thick and cleared mouse brain 136 μm from the tissue surface. Adapted from [26]. c Simultaneous labeling of mRNA transcripts and proteins using HCR and antibody staining (IHC) in uncleared tissue expressing endogenous fluorescent proteins (YFP). During the same period of time (18 h), HCR achieved uniform labeling of mRNA transcripts through the tissue section, whereas protein IHC only reached a shallow depth (~ 10 μm) below the surface (YZ view). The tissue section was soaked in refractive index matching solution for imaging up to 50 μm deep into the tissue. Magnified inserts: note a subset of cells show TH mRNA but not TH protein signal