Intravital staining to detect mineralization in Xenopus tropicalis during and after metamorphosis

Observing mineralization is essential for studying skeletal development, maintenance, and regeneration. Calcein and alizarin red have long been used to visualize mineralization in fixed specimens, but this requires the target animals to be sacrificed. However, several intravital bone-staining methods have been developed to visualize mineralized tissues in living animals. These methods have been applied to study fin rays and transparent fishes. Xenopus tropicalis is an excellent experimental animal model for studying bone formation and regeneration because skeletal mineralization begins during the free-living tadpole period, and its regenerative ability changes during metamorphosis. However, intravital bone staining of X. tropicalis has only been reported for tadpoles, and no details on its specificity or appropriate experimental conditions are available. Here, we compared the calcein- and alizarin red S (ARS)-staining methods and optimized these methods for tadpoles and juvenile frogs during and after metamorphosis. Staining with 0.01% ARS yielded acceptable signaling for young tadpoles, whereas calcein either at 0.1 or 0.01% occasionally showed artifactual staining of unmineralized tissues. In addition, 0.1% calcein or 0.1% ARS staining showed a higher signal-to-noise ratio with juvenile frogs compared to staining at 0.01%. We propose the use of 0.01% ARS for tadpoles before stage 61 and 0.1% ARS thereafter for staining mineralized tissues. Using this method, we found that ossification of the neural arches occurred at stage 51 in X. tropicalis. This method enables precise staging and manipulation based on the visualized bone structure.