The protocol given is designed for amphioxus embryos and larvae, obtained as described in Chapter 33. The method is modified from ref. 1 and has been used to visualize RNA from several developmentally expressed genes of amphioxus (see Note 1). With modification, it has also been successfully applied to embryos of other marine invertebrates, notably ascidia (e.g., ref. 2).

1. Fix amphioxus embryos and larvae (up to 3 d postfertilization) in freshly made 4% paraformaldehyde in MOPS buffer, at 4°C for 12 h.

2. After fixation, transfer specimens through two changes of 70% ethanol, and store at -20°C. Do not allow to freeze.

3. Digoxigenin-(DIG) labeled riboprobes are made by standard protocols (Chapter 42); redissolve probes in 50% formamide at 100 ng/|L, and store at -20°C. (see Notes 2 and 5).

4. Transfer selected embryos into one or more wells of a disposable Nunclon four-well dish, and add 0.5 mL PBT. All subsequent incubation, wash, and hybridization steps are performed in the same Nunclon dish at room temperature, unless otherwise stated. Each change of solution is performed using a Gilson pipetman, while observing under a dissecting microscope to ensure that embryos are not lost.

5. Change solution for fresh PBT.

6. Change solution for 7.5 |g/mL proteinase K in PBT; incubate without agitation for 10 min.

7. Quickly wash with two changes of PBT. Leave second wash for 5 min.

8. Change solution for 4% paraformaldehyde in PBT; incubate without agitation for 1 h.

9. Wash with two changes of 0.1 M triethanolamine, 2-5 min each.

10. Remove solution from embryos. Quickly add 2.5 |L acetic anhydride to 1 mL 0.1 M triethanolamine in a microfuge tube, vortex, and add 300 ||L onto embryos. Incubate without agitation for 5 min.

11. Repeat step 10.

12. Wash with two changes of PBT, 2-5 min each.

13. Change solution for a 1:1 mix of PBT and hybridization buffer; incubate for 5 min.

14. Change solution for hybridization buffer; incubate for 10 min.

15. Change solution for hybridization buffer prewarmed to 60°C; incubate at 60°C for at least 1 h, with horizontal rotation. A hybridization oven is useful.

16. Add 2 |L of DIG-labeled riboprobe from step 3 to 200 |L hybridization buffer. Heat to 75°C for 2 min (see Note 3).

17. Remove solution on embryos, and replace with the heated and diluted probe. Seal sides of the multiwell dish with Parafilm, and place on foam microfuge rack floating in 70°C water bath for 2 min.

18. Move the dish to the shaking oven, and incubate overnight at 60°C with horizontal rotation.

19. Wash embryos in prewarmed solution A, 2 x 30 min at 60°C with agitation.

20. Wash embryos in prewarmed solution B, 2 x 30 min at 60°C with agitation.

21. Wash embryos in prewarmed solution C, 2 x 30 min at 60°C with agitation.

22. For more stringent washes, wash with solution D, 30 min at room temperature.

23. Wash with two changes of PBT, 5-10 min at room temperature (see Note 4).

24. Change solution for blocking buffer; incubate for 1 h at room temperature.

25. Change solution for preabsorbed 1:3000 anti-DIG fab fragments. Place on a rotating platform at room temperature for 10 min, and then incubate overnight at 4°C. Agitation is not necessary.

26. Carefully remove antibody solution from embryos. Store antibody frozen for reuse.

27. Wash embryos in PBT, 2x for 10 min at room temperature.

29. Wash embryos in APT buffer, 2x for 10 min at room temperature.

30. Wash embryos in APT buffer, 2x for 20 min at room temperature.

31. Replace solution with NBT/BCIP staining mix, and place in dark (e.g., cover in foil) until color develops (10 min to 48 h). If staining requires longer than overnight, change for fresh NBT/BCIP mix each day. Some recommend that this step be performed in wash glasses; we find no difference between glass and plastic dishes.

32. When all embryos have developed a consistent staining pattern, wash with two changes of PBT and fix in 4% formaldehyde in PBT for 1 h at room temperature.

33. Mount embryos on slides in 80% glycerol in PBT. For amphioxus embryos, support the coverslip with strips of autoclave tape stuck onto the slide.

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