PUBLICATION

Two-color fluorescent in situ hybridization using chromogenic substrates in zebrafish

Authors
Schumacher, J.A., Zhao, E.J., Kofron, M.J., Sumanas, S.
ID
ZDB-PUB-141114-4
Date
2014
Source
Biotechniques   57: 254-6 (Journal)
Registered Authors
Schumacher, Jennifer, Sumanas, Saulius
Keywords
BCIP, FISH, NBT, Vector Red, confocal, fluorescent in situ hybridization, two color, zebrafish
MeSH Terms
  • Animals
  • Chromogenic Compounds/analysis*
  • Embryo, Nonmammalian/chemistry*
  • Embryo, Nonmammalian/cytology
  • Embryo, Nonmammalian/embryology
  • In Situ Hybridization, Fluorescence/methods*
  • Microscopy, Fluorescence, Multiphoton/methods*
  • Zebrafish
PubMed
25391914 Full text @ Biotechniques
Abstract
Two-color fluorescent in situ hybridization (FISH) is a widely used technique for comparing relative gene expression patterns. Current two-color FISH protocols are not ideal for detecting weakly expressed transcripts or monitoring signal strength and background levels during the course of the reaction. Here we describe an improved FISH protocol using the conventional highly sensitive chromogenic substrates nitro blue tetrazolium (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) and Vector Red in zebrafish embryos. This protocol substantially improves on existing FISH techniques by combining the advantages of long reactivity of alkaline phosphatase, chromogenic monitoring of both developing reactions, and the ability to perform subsequent high-resolution fluorescent imaging. Although tested in zebrafish, a similar approach is expected to be applicable to ISH in any model organism.
Genes / Markers
Figures
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Expression
Phenotype
Mutations / Transgenics
Human Disease / Model
Sequence Targeting Reagents
Fish
Antibodies
Orthology
Engineered Foreign Genes
Mapping