1 2 3 4

13. Pipet off all but 1 ml of prehybridization buffer, then add 10 µl DIG labeled
riboprobe. Incubate overnight at 70ºC. Prepare wash solution 1 and incubate O/N at
70 C to allow it to come to temperature.


14. Rinse embryos with 5 ml solution 1 to remove probe. Wash embryos 3X at 70º C
with solution 1 (30 minutes each).

15. Wash embryos 3X at 70º C with solution 3 (30 minutes each).

16. Wash embryos 3X with TBST containing 2 mM levamisole (5 minutes each).
- Heat inactivate small quantity of chick embryo powder in 1 ml TBST by heating to
70ºC for 30 minutes, chill on ice.

17. Pre-block embryos in 5 ml TBST containing 10% heat inactivated sheep serum.
(Filter the blocking solution through a 0.2 micron syringe filter before using.)Leave
for 2.5 hours at room temperature. (This prevents nonspecific binding of antibodies.)

-Pre-incubate 0.5 ml anti-DIG Fab-AP conjugate in 1 ml TBST containing 1% sheep
serum and appproximately 0.3% heat inactivated chick embryo powder (1/2000 final
dilution). Rock at 4ºC. (To remove cross-reactivity of antibody for embryos).

18. Spin the pre-incubated antibody solution for 10 minutes in microfuge. Remove
supernatent and push it through a syringe filter . Remove pre-blocking solution from
the embryos and add the filtered antibody. Incubate embryos overnight at 4ºC.


19. Wash embryos 3X at room temperature with TBST (5 minutes each).

20. Washembryos several more times (1 hour - 30 hours each) at room temperature
with TBST.

21. Wash 3X (10 minutes each) with CT buffer. (This puts embryos into alkaline
phosphatase reaction buffer).

22. Incubate embryos 15 minutes to 2 hours with detection solution, depending on
probe. Keep in dark during incubation.

23. Leave embryos overnight in PBT at 4ºC.

24. To enhance color, ascend and descend in MeOH/PBT series (5 minutes per wash):
25%, 50%, 75%, 100%, 100%, 75%, 50%, 25%.
Store embryos in PBT at 4ºC.

25. (optional) Clear embryos by equilibrating in glycerol/CMFET (1:1) followed by
glycerol/CMFET (4:1).

@2004 Cebra-Thomas

Last Modified: 17 August 2004

[Lab Protocols | Students | Cebra-Thomas | Course | Links ]