1 2 3 4

TBST/2 mM levamisole (500 ml):

500 mL 1 X TBST 240 mg levamisole

Pre-blocking solution (10 ml):

9 ml TBST 1 ml heat inactivated sheep serum

Anti -DIG pre-incubation solution (1 ml):

99% TBST (1 ml) 1% sheep serum (10 µl) 0.3% heat-inactivated embryo powder 150 mM NaCl (3 ml 5 M NaCl) 100 mM Tris (5 ml 2 M Tris, pH 9.5 25 mM MgCl2(0.52 ml 4.9 M MgCl2) dH2O

CT (100 ml):

PVA-CT: Prepare a 10% (w/v) solution of Polyvinyl alcohol (MW 31-50 kDa, Aldrich Chemicals) in CT buffer. PVA will only go into solution upon heating on a stir plate with moderate stirring. (Do not allow the solution to come to a full boil)Allow to cool down to room temp before using. AP detection solution (50 ml):PVA-CT 225 ml NBT 165 ml BCIP BCIP/NBT: Gibco 8280SA 5-Bromo-4-chloro-3-Indolylphosphate p-Toluidine salt, 50 mg/ml in DMF Nitroblue Tetrazolium Chloride, 75 mg/ml in 70% DMF

CMFET (500 ml)

4 g NaCl 0.1 gKCl 0.58 gNa2HPO4 (anhyd.) 0.1 gKH2PO4 (anhyd) 0.1 gEDTA 0.5 ml Tween-20 bring to 500 ml with dH2O.