A novel protocol of whole mount in situ hybridization(WISH)and its primary application in nile tilapia

To investigate the expression pattern and function of genes expressed in the development and organogenesis of nile tilapia embryos,whole mount in situ hybridization(WISH)of nile tilapia embryo was established based on the commonly used protocols in zebrafish(Danio rerio)and striped bass(Morone saxatilis).However,the WISH protocols of zebrafish and striped bass are not completely viable for nile tilapia embryos because of their huge,opaque yolk with early pigment formation.Thus we optimized the protocol for its application in nile tilapia.Specifically,we improved the methods of removing the pigments by H₂O₂ and KOH,and enhanced tissue permeabilization in tilapia embryos using cold acetone instead of proteinase K,reduced the times of washing embryos after being incubated in probes or antibodies,and optimized the system of image collection and the storage of embryos post-WISH.Using the recombination activating gene 1(Rag1)of nile tilapia as the probe gene,the result of WISH in nile tilapia embryos showed that its expression pattern was highly conserved compared with zebrafish Rag1 and medaka(Oryzias latipes)Rag1.The embryos post-WISH were unbroken and the gene expression position was distinct.The results indicated that the protocol of WISH in nile tilapia was effective and entirely feasible.