Alizarin Red S and Toluidine Blue for Differentiating Adult or Embryonic Bone and Cartilage

This technic has been successfully employed by the author for staining, in toto, the bones and cartilage of mature specimens of Urodela and the developing bone and cartilage of the embryonic human, cat, pig and rat. The differential staining is accomplished by using a modification of Dawson's method of staining bone with alizarin red S following a toluidine blue solution specific for cartilage. Specimens are fixed in 10% formalin, stained one week in a solution of .25 g. of toluidine blue in 100 cc. of 70% alcohol, macerated 5 to 7 days in a 2% KOH solution, counterstained for 24 hours in a 0.001% solution of alizarin red S in 2% aqueous KOH, dehydrated in cellosolve and cleared in methyl salicylate. In the adult and embryonic forms thus treated the soft tissues are cleared while the osseous tissue is stained red, the cartilage blue.