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

PMID:32394381

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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

In situ HCR

Anatomic

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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