Multiplexed Quantitative In Situ Hybridization with Subcellular or Single-Molecule Resolution Within Whole-Mount Vertebrate Embryos: qHCR and dHCR Imaging (v3.0).
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Abstract

In situ hybridization based on the mechanism of hybridization chain reaction (HCR) enables multiplexed quantitative mRNA imaging in the anatomical context of whole-mount vertebrate embryos. Third-generation in situ HCR (v3.0) provides automatic background suppression throughout the protocol, dramatically enhancing performance and ease of use. In situ HCR v3.0 supports two quantitative imaging modes: (1) qHCR imaging for analog mRNA relative quantitation with subcellular resolution in an anatomical context and (2) dHCR imaging for digital mRNA absolute quantitation with single-molecule resolution in an anatomical context. Here, we provide protocols for qHCR and dHCR imaging in whole-mount zebrafish, chicken, and mouse embryos.

Keywords

Anatomic
In situ HCR
Accuracy
Autofluorescence
Automatic background suppression
Fluorescence in situ hybridization (FISH)
Hybridization chain reaction (HCR)
In situ HCR v3.0
Multiplexed
Precision
Quantitative
Signal amplification
Single-molecule imaging
Thick sample penetration
Whole-mount vertebrate embryos
dHCR imaging
mRNA imaging
qHCR imaging

MeSH terms

Animals
Chickens
Diagnostic Imaging
Embryo, Mammalian
Embryo, Nonmammalian
In Situ Hybridization
In Situ Hybridization, Fluorescence
Mice
RNA, Messenger
Zebrafish

Authors

Choi, Harry M T
Schwarzkopf, Maayan
Pierce, Niles A

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