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Alizarin Red S (Bone) and Alcian Blue (Cartilage) Staining of Cleared Skeletons


### Method This method works well for 17.5 day mouse embryos. All stains should be made up fresh. Fix embryos in 90% ethanol for at least 1 week, longer if possible. Prior to staining remove skin, and viscera, particularly the liver, kidneys and gut. 1. Add embryo to approx. 20mls alcian blue solution, leave for 3 days. The alcian blue may begin to precipitate out, just ignore this. - Rehydrate as follows: - 70% ETOH 2-3hrs - 70% ETOH 2-3hrs - 40% ETOH 2-3hrs - 15% ETOH 2-3hrs - H20 until embryo sinks. - *NOTE: All ETOH steps can be longer*. - Add embryo to a freshly prepared 1% KOH solution until the embryo is quite clear. 1-2 days. - Add embryo to alizarin red S/KOH solution, until bone is stained purple. 2-3 days. (This solution can be changed daily). - Rinse in 1% KOH. ~ 3 changes several hours each. - 20% Glycerol in 1% KOH 24hrs (swirl occasionally) - 50% Glycerol in 1% KOH (swirl occasionally) - 80% Glycerol in 1% KOH (swirl occasionally) - 100% Glycerol (swirl occasionally) - 100% Glycerol (swirl occasionally) - Specimens are stored in 100% Glycerol or embedded in Resin. ### Recipes: 1. Alcian Blue (20mls) - 2mgs alcian blue, dissolve in 0.8ml H20 add 16ml Absolute ETOH and 4mls glacial acetic acid. - Alizarin Red S - Dissolve 1mg of alizarin red S in 100ml of 1% KOH
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