title: Two-color fluorescence in situ hybridization using chromogenic substrates inXenopus.
abstract: Xenopusembryo yolk proteins present a serious barrier to fluorescence microscopy. Previously, in situ assays of gene transcripts in whole embryos was limited to the use of chromogenic alkaline phosphatase substrates, which restricted researchers' ability to gauge coexpression of transcripts. Here, we describe a modified in situ hybridization (ISH) protocol that uses fluorescent substrates and a novel yolk-clearing technique for simultaneous visualization of the expression of two genes in wholeXenopusembryos with high resolution. This protocol employs two well-known fluorescent substrates, nitro blue tetrazolium/5-bromo- 4-chloro-3-indolyl-phosphate (NBT/BCIP) and Vector Red, in a sequential dual in situ hybridization procedure. Subsequent clearing of the samples with refractive-index-matching solution (RIMS) renders the samples amenable to confocal microscopy, allowing imaging at sufficiently high resolution to discern coexpression of transcripts within individual cells.
authors.affiliation: Division of Neonatology and Pulmonary Biology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Zachary
authors.initials: Z
authors.lastname: Agricola
authors.affiliation: Division of Developmental Biology, Cincinnati Children's Hospital Medical Center, OH.
authors.forename: Sang-Wook
authors.initials: SW
authors.lastname: Cha
mesh_terms: D000818:Animals; D002863:Chromogenic Compounds / Q000032:analysis / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000737:chemistry / Q000378:metabolism; D005456:Fluorescent Dyes / Q000032:analysis / Q000378:metabolism; D017404:In Situ Hybridization, Fluorescence / Q000379:methods*; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014981:Xenopus / Q000378:metabolism*
chemical_list: D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D011994:Recombinant Proteins
keywords: BCIP; FISH; NBT; Vector Red; Xenopus; confocal; fluorescent in situ hybridization; two color