Differential in Toto Staining of Bone, Cartilage and Soft Tissues

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Abstract
The following method for staining bone and cartilage allows study of the gross cleared specimen and does not injure the tissues for subsequent microscopic study: Fix in 10% neutral formalin; bleach thoroughly in 3% H2O2 in sunlight. Wash in distilled water. Stain bone 24 hours in 0.01 g. of Biebrich scarlet in 100 ml. of distilled water. Destain in 95% alcohol until soft tissues and cartilage are colorless. Stain cartilage 24 hours in a pH2 buffer solution of 2.1g. of citric acid per 100 ml. of water with 0.001 g. of methylene blue. Destain in pH2 buffer solution until soft tissues are pale. Dehydrate in two changes of 95% alcohol in preparation for clearing. (This completes the destaining and may remove too much stain from the cartilage if destaining in the pH2 solution has been carried too far.) Place in Groat's clearing fluid and cover loosely so that the alcohol may evaporate, or remove the alcohol in vacuo. Groat's Mixture No. 19 is usually satisfactory. For a combined stain, first stain bone, as above, and then apply the cartilage stain. Seal jars with an ordinary liquid wood glue such as LePage's.