Live Visualization of Calcified Bones in Zebrafish and Medaka Larvae and Juveniles Using Calcein and Alizarin Red S

Zebrafish and medaka are valuable model vertebrates for genetic studies. The advent of CRISPR-Cas9 technology has greatly enhanced our capability to produce specific gene mutants in zebrafish and medaka. Analyzing the phenotypes of these mutants is essential for elucidating gene function, though such analyses often yield unexpected results. Consequently, providing researchers with accessible and cost-effective phenotype analysis methods is crucial. A prevalent technique for investigating calcified bone development in these species involves using transgenic fish that express fluorescent proteins labeling calcified bones; however, acquiring these fish and isolating appropriate crosses can be time-consuming. We present a comprehensive protocol for visualizing ossified bones in zebrafish and medaka larvae and juveniles using calcein and alizarin red S staining, which is both economical and efficient. This method, applicable to live specimens during the ossification of bones, avoids apparent alterations in skeletal morphology and allows for the use of different fluorescent dyes in conjunction with transgenic labeling, thus enhancing the analysis of developmental processes in calcifying bones, such as vertebrae and fin rays. Key features • The calcified bones of alive zebrafish and medaka larvae and juveniles can be visualized repeatedly using simple and inexpensive calcein and alizarin red S. • No need to use transgenic fish to visualize ossified bones, allowing for rapid analysis of bone phenotypes in mutants. • Double staining is possible in transgenic fish with reporter genes such as GFP and DsRed using alizarin red S or calcein, which exhibit different fluorescence. • Ossification processes of bones such as vertebrae, ribs, and fin rays can be analyzed in mutants.