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This material is from the 4th edition of The Zebrafish Book. The 5th edition is available in print and within the ZFIN Protocol Wiki.
Molecular Methods

CHAPTER 9 - MOLECULAR METHODS

High Resolution whole-mount in situ hybridization

(Source: C. Thisse and B. Thisse)

Preparation of Probe:

Note: Work has to be done using gloves and sterile tubes and buffers.

1. Prepare DNA:

2. Synthesis of the antisense RNA probe. Incubate 2h at 37°C in transcription mix:

Transcription mix:

3. Digest the template DNA by adding 10 µl RNAse free DNAse for 15min at 37°C.

4. Stop the synthesis reaction and precipitate the RNA for 30 min with:

5. Centrifuge at 4°C for 30min at 12,OOO rpm

6. Wash with 70% ethanol, dry and resuspend in 20 µl sterile DEPC water.

7. Test 1 µl on agarose gel (generally 1 µl will be used for the hybridization).

Fixation and storage of embryos:

1. Remove chorions by pronase treatment (for embryos older than 18 somites) or manually (for earlier stages).

2. Fix embryos in 4% paraformaldehyde (PFA) in PBS overnight at 4°C.

3. Transfer embryos into 100% Methanol (MeOH), store them at -20°C (2h-several months).

In situ Day 1:

1. Rehydration: Transfer embryos into small baskets and rehydrate by successive incubtions in:

2. Digest with Proteinase K (10 µg/ml).

3. Refix in 4% PFA-PBS, 20 min.

4. Wash in PBT, 5 x 5 min.

5. Preadsorb the anti-DIG antibody (Boehringer) in a 1:1000 dilution in PBT-sheep serum 2%-BSA (2mg/ml) for several hours at RT with a batch of previously fixed embryos. Use about 500 embryos for 10 ml of antibody.

6. Prepare the Prehybridization and Hybridization mix:

Prehyb and Hybridization mix (HM):

Note: Add tRNA and Heparin to the prehybridation and hybridization only (not the wash solutions). Vary the % of formamide according to the desired stringency.

7. Prehybridize embryos in 800 µl of hybridization mix, 2 to 5 hrs at 70°C.

8. Remove prehybridization mix, discard, and replace with 200 µl of hybridization mix containing 100 - 200 ng of antisense RNA probe. Hybridize overnight in a waterbath at 70°C.

In situ Day 2:

Washes:

1. 100% HM at 70°C, very brief wash

2. 75% HM/25% 2 x SSC at 70°C, 15 min

3. 50% HM/50% 2 x SSC at 70°C, 15 min

4. 25% HM/75% 2 x SSC at 70°C, 15 min

5. 2 x SSC at 70°C, 15 min

6. Two washes of 30 min each in 0.2 x SSC if you hybridized on day one with 50% formamide or two washes of 30 min each with 0.05 x SSC if you hybridized with 65% formamide on day one.

7. 75% 0.2 (or 0.05) x SSC/25% PBT at RT, 10 min

8. 50% 0.2 (or 0.05) x SSC/50% PBT at RT, 10 min

9. 25% 0.2 (or 0.05) x SSC/75% PBT at RT, 10 min

10. PBT at RT, 10 min

11. PBT/2% sheep serum/2mg:ml BSA at RT, several hrs

Incubation with anti-DIG antiserum:

Incubate in antibody solution overnight with agitation at +4°C.

Anti-DIG antibody solution:

In situ Day 3:

Washes:

Remove antiserum, discard, and then wash extensively:

1. PBT at RT, very brief wash

2. PBT at RT, 6 x 15 min

3. Staining buffer (100 mM tris HCl pH9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% tween 20), 3 x 5 min

Staining:

1. Inclubate embryos in staining solution at RT and monitor with a dissecting microscope.

Staining solution:

(NBT stock: 50 mg Nitro Blue Tetrazolium in 0.7 ml of Dimethylformamide anhydre + 0.3 ml H2O. BCIP stock: 50 mg of 5-Bromo 4-Chloro3Indolyl Phosphate in 1 ml anhydrous Dimethylformamide).

2. Stop the reaction by removing the staining solution and washing the embryos in:

Stop solution:

3. Store the embryos in stop solution at +4°C in the dark.

Mounting:

1. For observation using a dissecting microscope, mount embryos directly in stop solution and methylcellulose.

2. For observation using a compound microscope, mount embryos in 100% glycerol.

3. For embryos at early developement stage (up to 18h), dehydrate in 100% methanol, clear for a few minutes in methylsalycilate, and mount in Permount.

Materials and supplies:

This protocol is adapted from:

Thisse, C., Thisse, B., Schilling, T. F., and Postlethwait, J. H. (1993). Structure of the zebrafish snail1 gene and its expression in wild-type, spadetail and no tail mutant embryos. Development. 119, 1203-1215.

If there are any questions, comments, or suggestions to improve this protocol, please contact us:

Christine Thisse and Bernard Thisse
IGBMC
1 rue Laurent Fries
67404 Illkirch Cedex, France
thisse@igbmc.u-strasbg.fr
The Zebrafish Book