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Methods Mol Biol. 2013;947:215-21. doi: 10.1007/978-1-62703-131-8_17.

Skeletal examination by double staining for ossified bone and cartilaginous tissue.

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  • 1BASF SE, Ludwigshafen, Germany.


The assessment of developmental toxicology data is a critical aspect of hazard evaluation for pharmaceuticals and environmental chemicals. Skeletal examination is an essential part of prenatal developmental toxicity studies of chemicals as well as pesticides and comprises evaluation of both cartilaginous and ossified skeletal components. Various techniques are published in the literature to process and double-stain skeletons of common laboratory animals which are all based on staining of the cartilage with Alcian Blue and staining of ossified bones with Alizarin Red S along with maceration (clearing) of the surrounding soft tissue. The staining of the cartilage allows the examination to assess ossified structures and their underlying cartilage in a single step.

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