Accurate whole-mount bone and cartilage staining requires acid-free conditions.

Bone and cartilage staining has provided anatomists with the ability to generate detailed descriptions of the adult and developing skeleton. Typically, Alizarin red S and Alcian blue are used for the staining of bone and cartilage, respectively. The binding of Alizarin red S and calcium is most stable at basic conditions, however, Alcian blue exhibits specific binding to polyanionic substances such as mucopolysaccharides under acidic conditions. Typical bone and cartilage staining protocols are conducted under acidic conditions. Because of this discrepancy in optimal pH, issues can arise in the staining of small specimens such as larval fish. Specifically, staining embryonic or larval specimens under acidic conditions can cause decalcification of small bones. Decalcification can completely inhibit the uptake of Alizarin red S in small bones. In order to mitigate this issue, researchers have developed an acid-free staining protocol that utilizes the concept of critical electrolyte concentration. While many researchers have adopted acid-free bone and cartilage staining, some researchers continue to stain these small specimens with acidic staining protocols. To ensure the reliability and validity of our skeletal descriptions, we urge scientists to utilize acid-free staining protocols when analyzing the skeletons of larval or embryonic specimens.