Whole-mount in situ hybridization (WISH) is a fundamental tool used for studying the spatio-temporal expression pattern of RNA molecules in intact embryos and tissues. The available methodologies for detecting mRNAs in embryos rely on enzymatic activities and chemical reactions that generate diffusible products, which are not fixed to the detected RNA, thereby reducing the spatial resolution of the technique. In addition, current WISH techniques are time-consuming and are usually not combined with methods reporting on the expression of protein molecules.
The protocol we present here is based on RNAscope® Technology allowing a robust and rapid method for visualization of multiple transcripts in zebrabfish embryos.
Azadeh Paksa & Theresa Gross-Thebing from Prof. Dr. Erez Raz's lab
Institute of Cell Biology - Center for Molecular Biology of Inflammation
Simultaneous high-resolution detection of multiple transcripts combined with localization of proteins in whole-mount embryos
Gross-Thebing T, Paksa A, Raz E.
BMC Biol. 2014 Aug 15;12(1):55.
Attendees will learn:
- How RNAscope® is used for the visualization of multiple transcripts demonstrated for three different RNA molecules in the 3-dimensional context of the developing embryo
- How to optimize the procedure allowing the preservation of embryo integrity, while exhibiting excellent signal-to-noise ratios
- How this method preserves the function of fluorescent proteins that are expressed in specific cells or cellular organelles and conserves antigenicity
- Which advantages RNAscope® offers compared to conventional in situ hybridization techniques