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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.
    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.

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.

Disclaimer: The following set of protocols were contributed by various members of our lab (past and present): Christine Andrews, Fiona Christensen, Neil Della, Ross Dickins, Debbie Donald, Andrew Holloway, Gary Hime, Colin House, Yinling Hu, Rachael Parkinson, Nadia Traficante, Hannah Robertson, Ping Fu and Dennis Wang. Special thanks to Vicki Hammond, Frank Kontgen and Maria Murphy, who contributed many of the ES cell protocols. Sections dealing with Photomicroscopy, Polyclonal and Monoclonal Antibody Production were provided by members of Gerry Rubin's Laboratory (Berkeley). Any comments in the methods (technical errors etc.) E-mail:
David Bowtell PMCI October 1998