Embryo preparation

1. Fix embryos in fresh 4% paraformaldehyde (or a thawed frozen aliquot) in PBS at 4° C overnight.
2. Dissect embryos (as needed) into PBT, and wash once in PBT for 5 minutes with gentle rocking.
3. Dehydrate embryos into methanol using a graded methanol/PBT series (25%, 50%, 75%, 100% methanol). Allow embryos to rock gently at room temperature for 5 minutes in each wash solution.
4. Do one additional wash in 100% methanol (5 minutes). At this point, embryos can be stored for up to 1 month (or more) at -20°C (use either glass scintillation vials or plastic screw-cap tubes).

Day 1: Prehybridization and Hybridization

1. Rehydrate samples in a 75%, 50%, 25% methanol/PBT series. Wash twice in PBT for 5 minutes at room temperature.
2. leach embryos (if necessary) with 6% hydrogen peroxide in PBT for 1 hour at room temperature with gentle rocking. (Note: bleaching time can be reduced or extended if desired.) Embryos must have been previously dehydrated in methanol if you are going to include the bleaching step!!
3. Wash 3 times with PBT for 5 minutes each.
4. Proteinase K treatment:
   Chick embryos: For younger embryos (< st 10), treat with 1 to 3 g/ml proteinase K in PBT for 15 minutes at room temperature. A similar treatment can be used for detecting ectodermal gene expression. For older embryos, (> st 10) a harsher treatment with proteinase K is necessary. Two parameters can be manipulated: the concentration of proteinase K and/or the length of treatment. Embryos younger than stage 18 are usually treated with 10 mg/ml proteinase K for about 15 minutes at room temperature. For embryos between stages 18 and 24, proteinase K treatment (10 mg/ml) can be extended for 20 to 25 minutes at room temperature. For stages 26 to 29, enzyme treatment with 10 mg/ml proteinase K can last for up to 40 minutes at room temperature. Alternatively, for embryos at stage 26 and older, the concentration of proteinase K can be increased while keeping the incubation time (15 minutes at room temperature) constant:
   stage: concentration of proteinase K
   26-27: 20 mg/ml
   28-29: 30 mg/ml
   30-31: 40 mg/ml
   32 and older: 50 mg/ml
   Mouse embryos:incubate in 10 mg/ml proteinase K in PBT for 5-10 minutes, depending on embryo stage. Use the following information as a guideline:
   embryo age: duration of incubation
   6.5 dpc: 4 min
   7.5 dpc: 4-5 min
   8.5 dpc: 6 min
   9.5 dpc: 10 min
   10.5 dpc: 15 min
   
5. Wash 10 minutes in 2 mg/ml glycine in PBT (make fresh).
6. Wash two times for 5 minutes each with PBT.
7. Postfix with 4% paraformaldehyde and 0.2% glutaraldehyde (0.2 ml of 25% stock per 25 ml) in PBT for 20 minutes at room temperature.
8. Wash 2 times for 5 minutes each in PBT.
   For mouse embryos, a slow equilibration in hybridization solution is suggested. This approach has not been routinely used for chick embryos, but it could probably be used successfully.
9. Wash 10 minutes in a 1:1 mixture of hybridization solution/PBT
10. Wash 10 minutes in hybridization solution.
11. Incubate at 70°C in hybridization solution for at least 1 hour.
12. Replace hybridization solution with fresh, add RNA probe (typically anywhere from one-fiftieth to one-tenth of a transcription reaction) and incubate overnight at 70° C.

1. Prewarm solution I to 70°C. Wash embryos 3 times for 30 minutes each at 70° C with prewarmed solution I .
2. Prewarm solution III to 65°C. Wash embryos 3 times for 30 minutes each at 65°C with prewarmed solution III.
3. Wash 3 times with fresh TBST for 5 minutes each at room temperature.
4. Blocking step:
   For chick embryos, block with 10% heat-inactivated sheep serum in TBST for at least one hour at room temperature.
   For mouse embryos, block embryos by incubating at room temperature for 60-90 minutes in blocking solution (10% heat-inactivated sheep serum and 0.1% Boerhinger Mannheim blocking reagent in TBST).
5. Preabsorb the antibody, if desired. (Note: A number of people do not preabsorb the antibody for chick embryos and still get good results, using a dilution of 1:5000 to 1:10,000).
   For chick embryos, put roughly 3 mg of chick embryo powder into a microtube with 0.5 ml TBST. Heat at 70° C for 30 minutes, vortex for 10 minutes. Cool on ice, add ml sheep serum and 1 m l anti-dig AP antibody. Shake gently at 4° C for 1 hr. Spin in microfuge for 10 minutes at 4° C. Collect supernatant into a 15 ml tube. Dilute with 1% sheep serum/TBST to a volume of 2 to 8 mls per vial of embryos, depending on the desired antibody dilution.
   For mouse embryos, follow the above procedure but use roughly 3 mg of mouse embryo powder and 0.5 ml TBST plus Boerhinger blocking reagent.

6. Remove blocking solution from embryos. Add antibody and incubate overnight at 4°C.

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