Posthybridization Washes

1. Solution 1: 50% (v/v) formamide, 5X SSC pH 4.5, 1% (w/v) SDS.

2. Solution 2: 0.5 M NaCl, 10 mM Tris-HCl, pH 7.5, 0.1 % (v/v) Tween-20.

3. RNase A (Boehringer Mannheim) 20 mg/mL in double-distilled water and stored in aliquots at -20°C.

4. Solution 3: 50% (v/v) formamide, 2X SSC pH 4.5 (pH using citric acid).

5. 10X TBST: 1.4 M NaCl, 27 mM KCl, 0.25 M Tris-HCl, pH 7.5, 1% (v/v) Tween-20. Dilute to 1X and add levamisole (Sigma) to 2 mM (24 mg/50 mL) on day of use.

6. Goat serum (Sigma) heat-inactivated at 55°C for 30 min and stored in 1-2 mL aliquots at -20°C. Heat-inactivated sheep serum can also be used.

7. Embryo powder: Homogenize 10-d chick embryos or 14-d mouse embryos in minimum volume of PBS, add 4 vol of ice-cold acetone, mix, and place on ice for 30 min. Spin at 10,000g for 10 min, and remove supernatant. Wash pellet with ice-cold acetone, and spin again. Spread the pellet out, and grind it into a fine powder on a sheet of filter paper. Allow it to air-dry. Store tightly capped at 4°C.

8. Anti-DIG or anti-FITC Fab fragments conjugated to alkaline phosphatase (Boehringer Mannheim).

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