Embryos can be processed either in small glass vials or in mesh-bottomed baskets. Using glass vials is more labor-intensive, since each vial must be handled individually and solution changes require aspiration and refilling of each. Baskets can be processed in mass, and only involves lifting the baskets from solution to solution. Additionally, the risk of damaging or losing the tissues is reduced when baskets are used. Baskets can be made by attaching small mesh to an Eppendorf tube from which the bottom has been removed. Alternatively, baskets are available commercially from Costar (15-mm Netwell, 74-|m mesh, cat. no. 3477) and fit easily into 12-well plastic dishes (Costar 12-well cluster, cat. no. 3513), which makes transferring the baskets to different solutions easy. A large number of embryos, from different stages, can be processed simultaneously for each probe of interest. A sense control hybridization should be included to control for nonspecific signal and overall level of background staining. Embryos can be transferred by using a Pasteur pipet from which the end has been cut to increase the size of the opening. Each set of embryos is then processed as follows:

1. Rehydrate embryos by incubating for 2-5 min in 100% Methanol (MeOH), 75% MeOH/25% H2O, 50% Me0H/50% H2O, 25% MeOH/75% 1X PBS + 0.1% Tween-20 (PBST), and 100% PBST.

2. Wash three times for 5 min in PBST.

3. Incubate embryos in 10 |g/mL Proteinase K in PBST. The time must be determined for each batch of Proteinase K, but a good starting point is approx 30 min at room temperature. This treatment is optional, but it probably increases sensitivity to some degree.

4. Rinse twice for 5 min in 0.1 M triethanolamine (pH 7.0-8.0).

5. Add 12.5 |L acetic anhydride/5 mL of triethanolamine and incubate for 5 min. Then, add another 12.5 |L of acetic anhydride and incubate for an additional 5 min. These steps block positively charged groups within the tissues (6).

6. Wash twice for 5 min in PBST.

7. Refix embryos by incubating in 4% paraformaldehyde in PBST for 20 min.

8. Wash five times for 5 min in PBST.

9. Replace PBST with hybridization buffer (listed below). If 5-mL glass vials are used, 500 |L are a sufficient volume to cover a large number of embryos. If baskets are used, an appropriate volume to cover all embryos must be used. For the Costar netwells, use 2-2.5 mL. Hybridization buffer:

50% Formamide, 5X SSC, 1 mg/mL Torula RNA, 100 |g/mL Heparin, 1X Denhardt's, 0.1% Tween-20, 0.1% CHAPS, and 5 mM EDTA.

10. After embryos settle through the dense hybridization solution, replace the buffer with fresh hybridization buffer and incubate at 60°C. Prehybridize embryos 5-6 h.

11. After prehybridization, replace the solution with hybridization buffer containing the probe (1 | g/mL). Hybridize overnight at 60°C.

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