Genetics and Genomics

scientificprotocols authored about 3 years ago

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.
  2. 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.
  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.
    • 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.
  2. Alizarin Red S
    • Dissolve 1mg of alizarin red S in 100ml of 1% KOH

DOI

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