230
HIDEYO OHUCHI AND SUMIHARE NO]I
the number of Hamburger-Hamilton's stage of the embryos examined). Try at 37°C when stronger treatment is needed on larger and mesenchymal tissues.
3. Wash for 5 mins with 2mg/ml glycine in PBT.
Note: This step is for complete inactivation of proteinase K and
can be omitted.
4. Wash twice for 5 mins each with PBT.
5. Refix the embryos with fresh 0.2% glutaraldehyde/4% paraformaldehyde in PBT for 20 mins.
6. Rinse the embryos twice for 5 mins with PBT.
(Optional) Incubate the embryos in PBT at 70°C for 40 to
75 mins. Chill on ice.
Note: This step (heat treatment) is for inactivation of endogenous peroxidase and can be omitted in the case of early embryos.
7. (Optional; for embryos older than about 2 days) Bleach for
lhr in 6% H 20 2/PBT. Wash with PBT, three times for 5 mins.
8. Remove most of the liquid, add 1 ml prewarmed prehybridization solution and allow the embryos to sink.
9. Replace with fresh 1 ml prehybridization solution and incubate at 65°C in hybridization oven for more than lh. The
embryos can be stored at -20°C at this point.
10. Add 700/Jl hybridization mix and incubate at 65°C overnight
with rocking.
Hybridization mix
Prehybridization buffer
Diluted probe (0.7-3.5 ng//ll )
Note: Cover the 24-well dish with Saran Wrap and put a water
tray in the oven to avoid drying up.
HIDEYO OHUCHI AND SUMIHARE NO]I
the number of Hamburger-Hamilton's stage of the embryos examined). Try at 37°C when stronger treatment is needed on larger and mesenchymal tissues.
3. Wash for 5 mins with 2mg/ml glycine in PBT.
Note: This step is for complete inactivation of proteinase K and
can be omitted.
4. Wash twice for 5 mins each with PBT.
5. Refix the embryos with fresh 0.2% glutaraldehyde/4% paraformaldehyde in PBT for 20 mins.
6. Rinse the embryos twice for 5 mins with PBT.
(Optional) Incubate the embryos in PBT at 70°C for 40 to
75 mins. Chill on ice.
Note: This step (heat treatment) is for inactivation of endogenous peroxidase and can be omitted in the case of early embryos.
7. (Optional; for embryos older than about 2 days) Bleach for
lhr in 6% H 20 2/PBT. Wash with PBT, three times for 5 mins.
8. Remove most of the liquid, add 1 ml prewarmed prehybridization solution and allow the embryos to sink.
9. Replace with fresh 1 ml prehybridization solution and incubate at 65°C in hybridization oven for more than lh. The
embryos can be stored at -20°C at this point.
10. Add 700/Jl hybridization mix and incubate at 65°C overnight
with rocking.
Hybridization mix
Prehybridization buffer
Diluted probe (0.7-3.5 ng//ll )
Note: Cover the 24-well dish with Saran Wrap and put a water
tray in the oven to avoid drying up.