Efficient and Easy-Dyeing Method of Whole-Mount Skeletal Staining

One of the most widely used methods for phenotypic analysis in developmental biology is whole-mount skeletal staining of mice. In this method, cartilage and bone are stained using two types of reagents, Alcian blue and Alizarin red, to observe the skeletal pattern of the whole body. Several experimental methods have been reported for whole-mount skeletal staining. Creating skeletal specimens that are clearly visible is possible using these methods. However, staining all high-quality samples to observe a large number of skeletal specimens takes considerable time and effort. Therefore, in this paper, we review and modify the conventional protocol and describe an efficient and simple experimental method that we use to observe the morphology of the vertebrae and digit patterns at the tips of the hands and feet, in a manner similar to other methods. We also explain the details of the experimental method for fabricating skeletal specimens using a conventional protocol and a unique potassium hydroxide (KOH) processing method. Herein, we present a simple method for the efficient fabrication of several skeletal specimens.