Figure 4.

The principles of in situ hybridization chain reaction. (A) visualization of hybridization chain reaction (HCR) where metastable fluorescent harpins assemble into amplification polymers for the detection of initiators; the presence of an initiator triggers hybridization chain reaction. H1 complex nucleates with hairpin H2, providing the basis for a chain reaction alternating polymerization steps. (B,C) Every HCR protocol consists of two stages: detection and amplification stage. In the detection stage probes are hybridized to the target mRNA sequences and nonhybridized material is washed from the sample. In the amplification stage specially banded probes trigger fluorescent amplification polymers and every material present in the reaction which does not bind is washed; these washing steps minimalize the presence of background signals and nonspecific signals. (D) On C is visualized an experimental timeline of the reaction with the exact length of time of each step [47].