Evicerate embryos in cold PBS.
Fix in 0.2% para in 0.1M PIPES for 1 hour on coldroom nutator (4°C).
Rinse with cold PBS on coldroom nutator 5 min.
Rinse with Detergent Free Buffer (DFB) 3 x 30 min. at room temp. on nutator.
In 2ml DFB add:
20µl Ferricyanide (5mM final)
20µl Ferrocyanide (5mM final)
100µl X-Gal (20mg/ml) (1mg/ml final)
Incubate @ 37°C O/N in dark.
Stop reaction with PBS (Ca/Mg+)
50ml 0.2% para in 0.1M PIPES
22.5ml H2O + 2 drops NaOH
HEAT
Add 0.1g para, dissolve, place on ice.
Add 25ml 0.2M PIPES pH6.9
2.5ml 0.1M EGTA
100µl 1M MgCl2
Sterile filter, keep on ice.
0.2M PIPES pH 6.9
3.35g up to 50ml with H2O
Detergent Free Buffer
0.1M PB
2mM MgC12
1% DMSO