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

Methods Mol Biol. 2020:2148:159-178. doi: 10.1007/978-1-0716-0623-0_10.

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: 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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Chickens
  • Diagnostic Imaging / methods*
  • Embryo, Mammalian
  • Embryo, Nonmammalian
  • In Situ Hybridization / methods*
  • In Situ Hybridization, Fluorescence / methods*
  • Mice
  • RNA, Messenger / genetics*
  • Zebrafish / genetics

Substances

  • RNA, Messenger