Abstract
ABSTRACTRNA in situ hybridization based on the mechanism of the hybridization chain reaction (HCR) enables multiplexed, quantitative, high-resolution RNA imaging in highly autofluorescent samples, including whole-mount vertebrate embryos, thick brain slices and formalin-fixed paraffin-embedded tissue sections. Here, we extend the benefits of one-step, multiplexed, quantitative, isothermal, enzyme-free HCR signal amplification to immunohistochemistry, enabling accurate and precise protein relative quantitation with subcellular resolution in an anatomical context. Moreover, we provide a unified framework for simultaneous quantitative protein and RNA imaging with one-step HCR signal amplification performed for all target proteins and RNAs simultaneously.
Highlights
Biological circuits encoded in the genome of each organism direct development, maintain integrity in the face of attacks, control responses to environmental stimuli, and sometimes malfunction to cause disease
SUMMARY: Signal amplification based on the mechanism of hybridization chain reaction enables multiplexed, quantitative, highresolution imaging of protein and RNA targets in highly autofluorescent tissues
The probes for different targets are labeled with different hybridization chain reaction (HCR) initiators that trigger orthogonal HCR amplifiers labeled with spectrally distinct fluorophores
Summary
Biological circuits encoded in the genome of each organism direct development, maintain integrity in the face of attacks, control responses to environmental stimuli, and sometimes malfunction to cause disease. CARD is widely used despite three significant drawbacks: multiplexing is cumbersome due to the lack of orthogonal deposition chemistries, necessitating serial amplification for one target after another (Lehmann & Tautz, 1994; Nieto et al, 1996; Thisse et al, 2004; Denkers et al, 2004; Kosman et al, 2004; Clay & Ramakrishnan, 2005; Barroso-Chinea et al, 2007; Toth & Mezey, 2007; Acloque et al, 2008; Piette et al, 2008; Glass et al, 2009; Stack et al, 2014; Mitchell et al, 2014; Tsujikawa et al, 2017), staining is qualitative rather than quantitative, and spatial resolution is routinely
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