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(Source: C. Thisse and B. Thisse from Zebrafish Book 5th Edition)

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Preparation of Probe:

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

1. Prepare DNA:

ο Linearize 5 µg of DNA by digesting with the appropriate restriction enzyme for 2h.
ο Stop the reaction using first a mix of phenol/chloroform and then chloroform.
ο Precipitate the DNA with Ethanol, centrifuge, and wash with RNAse free 70% Ethanol.
ο Resuspend the DNA in 10 mM Tris and 1 mM EDTA.
ο Test an aliquot on Agarose gel.

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

Transcription mix:

1µg linearized DNA
Transcription buffer (T3 or T7 RNA polymerase) 4 µl
NTP DIG RNA (Boehringer) 2 µl
RNAse inhibitor (35 units/µl) 1 µl
T3/T7 RNA polymerase (20 units/µl, Stratagene) 1 µl
Sterile water to 20 µl total

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

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

ο 1 µl EDTA 0.5M pH 8
ο 2.5 µl LiCl 4M
ο 75 µl Ethanol 100% at 70°C

5. Centrifuge at 4°C for 30minutes at 12,000 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:

Rehydration: Transfer embryos into small baskets and rehydrate by successive incubations in:
75% MeOH 25% PBS for 5 minutes
50% MeOH 50% PBS for 5 minutes
25% MeOH 75% PBS for 5 minutes
100% PBT (PBS/Tween20 0.1%) 4 x 5 minutes

Digest with Proteinase K (10 µg/ml):
blastula and gastrula: 30 seconds
early somitogenesis: 1 minute
late somitogenesis (14 to 22 somites): 5 minutes
24 hpf embryos: 15 minutes
36/48 hpf embryos: 30 minutes

Refix in 4% PFA PBS, 20 minutes.

Wash in PBT, 5 x 5 minutes.

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

Prepare the Prehybridization and Hybridization mix:

Prehyb and Hybridization mix (HM):

ο Formamide 50 65%
ο 5 x SSC
ο Tween20 0.1%
ο Citric acid to pH 6.0 (460 µl of 1M for 50 ml)
ο Heparin 50 µg/ml
ο tRNA 500 µg/ml

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 hours 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 water bath 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 minutes

3. 50% HM/50% 2 x SSC at 70°C, 15 minutes

4. 25% HM/75% 2 x SSC at 70°C, 15 minutes

5. 2 x SSC at 70°C, 15 minutes

6. 0.2 x SSC, 50% formamide (for normal stringency) or 0.05 x SSC, 65% formamide (for high stringency), 2 x 30 minutes

7. 75% 0.2 (or 0.05) x SSC/25% PBT at room temperature, 10 minutes

8. 50% 0.2 (or 0.05) x SSC/50% PBT at room temperature, 10 minutes

9. 25% 0.2 (or 0.05) x SSC/75% PBT at room temperature, 10 minutes

10. PBT at room temperature, 10 minutes

11. PBT/2% sheep serum/2mg:ml BSA at room temperature, several hours

Incubation with anti DIG antiserum:

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

Anti DIG antibody solution:

Preabsorbed anti DIG, 1:5000 dilution (final concentration) in PBT
2% sheep serum
2mg/ml BSA

In situ Day 3:

Washes:

Remove antiserum, discard, and then wash extensively:

PBT at room temperature, very brief wash

PBT at room temperature, 6 x 15 minutes

Staining buffer (100 mM Tris HCl pH9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% Tween 20), 3 x 5 minutes

Staining:

1. Incubate embryos in staining solution at room temperature and monitor with a dissecting microscope.

Staining solution:

NBT 50 mg/ml 225 µl
BCIP 50 mg/ml 175 µl
Staining buffer 50 ml

NBT stock:

50 mg Nitro Blue Tetrazolium in 0.7 ml of Dimethylformamide anhydrous + 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:

PBS pH5.5
EDTA 1mM

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 development stage (up to 18h), dehydrate in 100% methanol, clear for a few minutes in methyl salicylate, and mount in Permount.

Materials and supplies:

ο PFA: paraformaldehyde (Sigma)
ο 10 x PBS
ο MeOH: methanol
ο Tween20 (Sigma P1379)
ο Proteinase K (Boehringer 1000144)
ο Anti DIG antibody alkaline phosphatase Fab fragment (Boehringer 1 093 274)
ο BSA fraction V protease free (Sigma A 3294)
ο Formamide (deionized, high purity grade)
ο 20 x SSC
ο Heparin at 5 mg/ml (Sigma H3393)
ο RNAse free tRNA (Sigma R7876, 50 mg/ml resuspended in H2O and extensively extracted several times in Phenol/CHCl3 to remove protein)
ο Citric acid 1M
ο Normal Sheep serum (Jackson Immunoresearch 013 000 121)
ο Tris HCl pH9.5 1M
ο MgCl2 1M
ο NaCl 5M
ο NBT 50 mg/ml (made from powder, Sigma N6876)
ο BCIP 50 mg/ml (made from powder, Sigma B8503)
ο PBS pH5.5
ο EDTA 0.5M
ο Glycerol 100%
ο Methyl salicylate (Sigma M6752)
ο Permount (Fisher SP15 100)

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:

Christine Thisse and Bernard Thisse
IGBMC, 1 rue Laurent Fries
67404 Illkirch Cedex
FRANCE
thisse@igbmc.u strasbg.fr