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:
- Linearize 5 µg of DNA by digesting with the appropriate restriction enzyme for 2h.
- Stop the reaction using first a mix of phenol/chloroform and then chloroform.
- Precipitate the DNA with Ethanol, centrifuge, and wash with RNAse free 70% Ethanol.
- Resuspend the DNA in 10 mM Tris and 1 mM EDTA.
- 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 15min at 37°C.
4. Stop the synthesis reaction and precipitate the RNA for 30 min with:
- 1 µl EDTA 0.5M pH 8
- 2.5 µl LiCl 4M
- 75 µl Ethanol 100% at -70°C
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:
- 75% MeOH - 25% PBS for 5 min
- 50% MeOH - 50% PBS for 5 min
- 25% MeOH - 75% PBS for 5 min
- 100% PBT (PBS/Tween20 0.1%) 4 x 5 min
2. Digest with Proteinase K (10 µg/ml).
- blastula and gastrula: 30 seconds
- early somitogenesis: 1 min
- late somitogenesis (14 to 22 somites): 5 min
- 24h embryos: 15 min
- 36h/48h embryos: 30 min
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):
- Formamide 50-65%
- 5 x SSC
- Tween20 0.1%
- Citric acid to pH 6.0 (460 µl of 1M for 50 ml)
- Heparin 50 µg/ml
- 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 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:
- Preadsorbed 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:
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 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
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:
- PFA: paraformaldehyde (Sigma)
- 10 x PBS
- MeOH: methanol
- Tween20 (Sigma P1379)
- Proteinase K (Boehringer 1000144)
- Anti DIG antibody - alkaline phosphatase Fab fragment (Boehringer 1 093 274)
- BSA fraction V protease free (Sigma A-3294)
- Formamide (deionized, high purity grade)
- 20 x SSC
- Heparin at 5 mg/ml (Sigma H3393)
- RNAse free tRNA (Sigma R7876, 50 mg/ml resuspended in H2O and extensively extracted several times in Phenol/CHCl3 to remove protein)
- Citric acid 1M
- Normal Sheep serum (Jackson Immunresearch 013-000-121)
- Tris HCl pH9.5 1M
- MgCl2 1M
- NaCl 5M
- NBT 50 mg/ml (made from powder, Sigma N6876)
- BCIP 50 mg/ml (made from powder, Sigma B8503)
- PBS pH5.5
- EDTA 0.5M
- Glycerol 100%
- Methylsalycilate (Sigma M6752)
- 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 us:
Christine Thisse and Bernard Thisse
IGBMC
1 rue Laurent Fries
67404 Illkirch Cedex, France
thisse@igbmc.u-strasbg.fr
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