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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.
NB: 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) 20% Glycerol in 1% KOH 24hrs (swirl occasionally)
50% Glycerol in 1% KOH "
80% Glycerol in 1% KOH "
100% Glycerol "
100% Glycerol "
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.