1. Dissect out embryos (see Note 2) in 1X PBS (mice) or Howard's Ringer (chicks).
2. Fix in a large volume (at least five times the volume of embryonic tissue) of 4% w/v parafomaldehyde in PBS at 4°C overnight in a vessel that adequately contains the fumes (see Note 3). Smaller embryos can be fixed for shorter periods of time (4 h minimum), and it is convenient to dechorionate zebrafish after fixation.
3. Wash twice in PBT (for this and subsequent procedures up to hybridization step 14 use an excess [e.g., 10-fold by volume] of solution over embryo tissue).
4. Embryos are then dehydrated in a graded methanol series: this dissolves membranes and helps to prevent gas bubbles from forming during the subsequent bleaching step. Wash with 25% methanol/75% PBT, and then 50% methanol/ 50% PBT, then 75% methanol/25% PBT each for 3-5 min with rocking. Finally, wash with 100% methanol twice each for 30 min (see Note 4).
5. Rehydrate embryos by washing with 75% methanol/25% PBT, then 50% methanol/50% PBT, and then 25% methanol/75% PBT each for 5-10 min with rocking. Finally, wash twice with PBT each for 5 min.
6. Bleach embryos with 6% (v/v) hydrogen peroxide in PBT for 1 h.
8. Treat with 10 | g/mL proteinase K in PBT for an appropriate amount of time (see Notes 5 and 6) e.g., for chick embryos: 5-7 min for HH stage 10, 10 min for HH stage 17, and 20 min for HH stage 22 and above).
9. Rinse twice with PBT for 5-10 min (depending on age).
10. Postfix using 0.2% (w/v) gluteraldehyde, and 4% (w/v) paraformaldehyde in PBS for 20 min in the fume hood.
11. Wash three times with PBT.
12. Remove PBT, and rinse embryos once with prewarmed prehybridization solution.
13. Replace prehybridization solution with fresh prehybridization solution, and incubate at 70°C for at least 1 h (see Note 7).
14. Remove prehybridization solution and replace with prewarmed hybridization solution containing approx 1 |g/mL of riboprobe (usually 10 |L probe to 1 mL prehybridization solution; see Subheading 3.4.). For "two-color" in situ's, add both DIG- and FITC-labeled probes simultaneously. Incubate at 70°C overnight. Use at least sufficient hybridization solution to cover the embryos completely.
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