Fluorescent In Situ Hybridization in Embryos of the African Turquoise Killifish Nothobranchius furzeri

Abstract

Precisely where and when a given gene is expressed is crucial for our understanding of developmental and cell biology but determining this is often constrained by detection limits. Here, we describe a technique for visualization of low-copy mRNA in Nothobranchius furzeri embryos using tyramide signal amplification (TSA). In this protocol, an anti-sense digoxigenin-labeled RNA probe is hybridized to mRNA in situ. Anti-digoxigenin antibodies conjugated to horseradish peroxidase (POD) are then bound to the probe and reacted with fluorescently labeled tyramide. Combining this method with a counterstain, such as DAPI, allows for the detection of mRNA at a single-cell resolution.