Two-color fluorescent in situ hybridization using chromogenic substrates in zebrafish.
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.
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Jennifer A
JA
Schumacher
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Emma J
EJ
Zhao
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Matthew J
MJ
Kofron
Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, OH.
Saulius
S
Sumanas
D000818:Animals; D002863:Chromogenic Compounds / Q000032:analysis*; D004625:Embryo, Nonmammalian / Q000737:chemistry* / Q000166:cytology / Q000196:embryology; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D036641:Microscopy, Fluorescence, Multiphoton / Q000379:methods*; D015027:Zebrafish
D002863:Chromogenic Compounds
BCIP; FISH; NBT; Vector Red; confocal; fluorescent in situ hybridization; two color; zebrafish