Here we propose a new protocol for whole-mount bone staining, which allows the rapid preparation of highly cleared and nondestructive specimens. It only takes 3 days to complete whole procedure for small vertebrates, such as medaka, zebrafish, and Xenopus frogs. In this procedure, we used a newly developed fixative containing formalin, Triton X-100, and potassium hydroxide, which allows the fixation, decolorization, and transparentization of specimens at the same time. A bone staining solution containing alizarin red S with ethylene glycol and a clearing solution containing Tween 20 and potassium hydroxide also contributed the specificity and swiftness of this new system. As expected, although details of the skeletal system could be observed in specimens with high transparency, it was noteworthy that high-resolution fluorescence images acquired using zoom microscopes clearly delineated the shape of each bone. This new procedure would be expected to be widely used as a standard procedure for bone staining in the testing the developmental toxicity of chemicals and in the screening test of knockout or mutant animals.

In this Article, the legend of Figure 3 is incorrect: "A flowchart of the rapid (RAP) system for bone staining (RAP-B). The new procedure for bone staining which is rapid, nondestructive, and allowed to obtain high-definition, fine bone-stained specimens, was based on our RAP system. Cartilage staining (RAP-C) can be applied as single staining or optionally added before the bone staining for double staining of bone and cartilage (RAP-B/C, Supplementary Fig. S8). FIXATIVE: 5% formalin, 5% Triton X-100, 1% potassium hydroxide (KOH); B-STAINING SOLUTION: 0.05% alizarin red S, 20% ethylene glycol, 1% KOH; C-STAINING SOLUTION: 50-70% ethanol, 20% acetate, 0.015-0.02% alcian blue; CLEARING SOLUTION: 20% Tween 20, 1% KOH; ENHANCEMENT SOLUTION: 20% ethylene glycol, 5% Triton X-100, 1% KOH. " should read: "A flowchart of the rapid (RAP) system for bone staining (RAP-B). The new procedure for bone staining which is rapid, nondestructive, and allowed to obtain high-definition, fine bone-stained specimens, was based on our RAP system. Cartilage staining (RAP-C) can be applied as single staining or optionally added before the bone staining for double staining of bone and cartilage (RAP-B/C