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

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.
2. Rehydrate as follows:
   1. 70% ETOH 2-3hrs
   2. 70% ETOH 2-3hrs
   3. 40% ETOH 2-3hrs
   4. 15% ETOH 2-3hrs
   5. H20 until embryo sinks.

      NOTE: All ETOH steps can be longer.

3. Add embryo to a freshly prepared 1% KOH solution until the embryo is quite clear. 1-2 days.
4. Add embryo to alizarin red S/KOH solution, until bone is stained purple. 2-3 days. (This solution can be changed daily).
5. Rinse in 1% KOH. ~ 3 changes several hours each.
6. 1. 20% Glycerol in 1% KOH 24hrs (swirl occasionally)
   2. 50% Glycerol in 1% KOH (swirl occasionally)
   3. 80% Glycerol in 1% KOH (swirl occasionally)
   4. 100% Glycerol (swirl occasionally)
   5. 100% Glycerol (swirl occasionally)
   6. Specimens are stored in 100% Glycerol or embedded in Resin.

2mgs alcian blue, dissolve in 0.8ml H20 add 16ml Absolute ETOH and 4mls glacial acetic acid.

Dissolve 1mg of alizarin red S in 100ml of 1% KOH.