Hematopoietic and Vascular System Toxicity - Zebrafish: Methods for Assessing Drug Safety and Toxicity - page 79

2. Purify the DNA.
3. Perform an RNA polymerase reaction.
4. Treat with DNase I.
5. Purify RNA probe and maintain at 80 C.

In Situ Hybridization: Embryos (Thisse and Thisse, 2008)

1. Embryos are fixed at specific stages in development in 4% PFA at 4 C
overnight. If the embryos have developed pigment (are older than 24hpf),
then bleaching is required. If not, skip to step 5.
2. Wash embryos 1

in PBS with 0.1% Tween (PBT).
3. Incubate at room temperature in a 3% H 2 O 2 /0.8% KOH solution until the
pigment has disappeared. This will take from 1 to 20min, depending on
embryo stage.
4. Wash embryos 1 in PBT for 5min.

5. Refix in 4% PFA for at least 20min.
6. Wash 1-2

for 5 min each in PBT.
7. Dehydrate embryos in 100% methanol for 3 at 5min each at room

temperature.
8. Incubate embryos for at least 2 h at

20 C. Alternatively, they can be kept at

this temperature for several months.
9. Rehydrate embryos into PBT by 5min incubations in the following solu-
tions: 75% MeOH in PBS, 50%, 25%, and 100% PBS.
10. Wash embryos 4

for 5 min each in PBT.
11. Permeabilize with proteinase K (10 mg/mL) at room temperature. Length of
this incubation is dependent on embryo stage, and detailed information can
be found in Thisse and Thisse (2008).
12. After this reaction is stopped, refix embryos in PFA for at least 20min.
13. Wash embryos 4 for 5 min each in PBT.

14. Incubate for 2-5 h at 70 C in prehybridization buffer (50% formamide, 5

SSC, 0.5mg/mL torula yeast RNA, 50mg/mL heparin, 0.1% Tween in
sterilized water).
15. Dilute probe 0.8-1.5 ng/ L in prehybridization buffer.

16. Incubate embryos in probe overnight at 70 C.
17. Remove probe and gradually change to 2

m

SSC by the following 15min

washes at 70 C: 75% Hybe in 2

SSC, 50%, 25%, and 100% 2 SSC.

SSC at 70 C.
19. Gradually change to PBT by washing for 10 min in the following solutions:
75% 0.2

18. Wash 2 for 30 min in 0.2

SSC in PBT, 50%, 25%, and 100% PBT.
20. Incubate for 3 h at room temperature in blocking buffer solution (100 mg
BSA and 1mL 100% lamb/sheep serum in 50mL PBT).
21. Add anti-dig antibody to blocking solution and incubate embryos overnight
at 4 C.
22. Wash embryos in PBT 6 for 15min each at room temperature shaking.