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

protocol.md

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