Materials

1. MOPS buffer: 0.5 M NaCl, 1 mM MgSO4, 2 mM EGTA, 0.1 M morpho-linopropanesulfonic acid buffer, pH 7.5. Sterile.

2. Paraformaldehyde: electron microscopy-grade (TAAB, Aldermastan, UK).

3. Nunclon four-well multiwell dishes (Nunc cat. no. 176740, Life Technologies, Paisley, Scotland).

4. PBS: 0.9 % (w/v) NaCl, 20 mM sodium phosphate buffer, pH 7.4. Sterile.

6. Proteinase K (10 mg/mL stock). Store frozen.

7. 0.1 M Triethanolamine, pH 8.0. Sterile.

8. Acetic anhydride.

9. Hybridization buffer: 50% formamide (Fluka, Gillingham, UK), 1.3X SSC, pH 4.5, 5 mM EDTA, pH 8.0, 500 ^g/mL yeast RNA, 0.2% (v/v) Tween 20, 0.5% CHAPS, 100 ^g/mL heparin, in DEPC-treated sterile water.

10. Oven with shaking platform, e.g., Hybaid Midi-Dual oven.

11. Wash solution A: 50% formamide (Fluka), 4X SSC, 0.1% Tween-20.

12. Wash solution B: 50% formamide (Fluka), 2X SSC, 0.1% Tween-20.

13. Wash solution C: 50% formamide (Fluka), 1X SSC, 0.1% Tween-20.

15. Blocking buffer: 5% (w/v) Boehringer Mannheim (Lewes, UK) digoxigenin blocking reagent, 2 mg/mL BSA in PBT. Takes over 1 h to dissolve at 70°C. Store frozen.

16. Amphioxus powder: Grind frozen adult amphioxus under liquid nitrogen, or homogenize in PBS. Then incubate in ice-cold acetone for 30 min. Recover powder by centrifugation, and grind further in a pestle and mortar. Store dry powder frozen.

17. Sheep serum: Heat to 55°C for 30 min. Store frozen.

18. Preabsorbed 1:3000 anti-DIG fab fragments: To 1.5 mg of amphioxus powder, add 400 ||L PBT, and heat to 70°C for 10 min. Add 50 ||L 20 mg/mL BSA, 50 ||L pretreated sheep serum, 0.5 ||L anti-DIG fab fragments (Boehringer Mannheim). Mix and incubate at 37°C for 1 h, and then overnight at 4°C. Centrifuge for 5 min to remove powder; to the supernatant, add 1 mL PBT containing 2 mg/mL BSA. Add 50 |L pretreated sheep serum. Store at -20°C in 200-|L aliquots.

19. APT buffer: 100 mM NaCl, 100 mM Tris-HCl, pH 9.5, 50 mM MgCl2, 1% Tween-20.

20. NBT/BCIP staining mix: Just before use, add 4.5 |L stock nitro blue tetrazolium salt (NBT) and 3.5 |L stock 5-bromo-4-chloro-3-indolyl phosphate (BCIP) to 1 mL APT buffer containing 2 mM levamisole. Stock NBT is 75 mg/mL in 70% dimethyl formamide, and stock BCIP is 50 mg/mL in 100% dimethyl formamide; both stocks are supplied by Gibco-BRL as Immunoselect kit.

21. 37-40% Formaldehyde stock solution.

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