Posthybridization Washes and Color Detection

1. Formamide-GPR (BDH/Merk, Poole, UK) Formamide is toxic. Avoid contact with the skin and use witin a fume cupboard.

4. Sheep serum (Sigma): store in aliquots at -20°C.

5. Alkaline-phosphatase anti-digoxygenin Fab fragments (Boehringer Mannheim, Lewes, UK). Store at 4°C.

6. Embryo Powder: Homogenise suitable tissue, i.e., tissue being analyzed, in a minimum vol of PBS. Add 4 vol of ice-cold acetone, mix and place on ice for 30 min. Spin at 10,000g for 10 min. Discard supernatant and wash pellet in ice-cold acetone and respin. Spread pellet on filter paper and grind to a fine powder. Allow to air dry. Store tightly capped at 4°C.

7. Blocking reagent for nucleic acid hybridization and detection (Boehringer Mannheim).

8. TBST: 10X TBS-135 mM NaCl, 250 mM Tris-HCl, pH 7.5. On day of use dilute to 1X and add 0.024 g levamisole and 0.5 mL Tween-20 to each 50 mL of 1X TBST.

9. Alkaline phosphatase buffer: 100 mM NaCl, 50 mM MgCl2, 100 mM Tris-HCl pH 9.5, 1% Tween-20. Make on the day from stock solutions.

10. Color solution is alkaline phosphatase buffer plus 45 pL/100 mL NBT (75 mg/mL, 4-nitro Blue Tetrazolium chloride, Boehringer Mannheim) and 35 pL/100 mL of

X-phosphate (50 mg/mL, 5-Bromo-4-chloro-3-indolyl-a-d-galacto-pyranose, Boehringer Mannheim), and 2 mM levamisole (Sigma). Levamisole is made fresh from solid every 2 wk and is stored at 4°C.

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