ZFIN ID: ZDB-PUB-141114-4
Two-color fluorescent in situ hybridization using chromogenic substrates in zebrafish
Schumacher, J.A., Zhao, E.J., Kofron, M.J., Sumanas, S.
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
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.