Whole-mount in situ hybridization
(Ch. Thisse and B. Thisse, IGBMC, 1 rue Laurent Fries, 67404 ILLKIRCH Cedex, FRANCE)
Work has to be done using sterile tubes and buffers and gloves
- DNA preparation :
5mg of DNA is linearized with the appropriate restriction enzyme during 2h. The reaction is then stopped using first a mix of phenol/chloroform, then chloroform. The DNA is then Ethanol precipitated, centrifuged and washed with RNAse free 70% Ethanol. DNA is then resuspended in Tris EDTA 10mM/1mM and an aliquot is tested on agarose gel.
Alternatively, inserts can be PCR amplified and the purified product used as template for the synthesis of the probe.
- RNA probe synthesis :
Transcription mix :
1mg linearized DNA
Transcription buffer (T3 ou T7 RNA polymerase) : 4ml
NTP-DIG-RNA (Boehringer) : 2ml
RNAse inhibitor (35u/ml) : 1ml
T3/T7 RNA polymerase (20u/ml, Stratagene) : 1ml
Sterile water : up to 20ml
- 2h at 37°C
- Digest the template DNA by adding 2ml RNAse free DNAse during 15mn at 37°C.
- Stop the synthesis reaction and precipitate the RNA with 1ml EDTA 0.5M pH 8, 2.5ml LiCl 4M and 75ml cold Ethanol 100%. After storage at-70°C during 30mn, centrifuge at 4°C, during 30mn at 12OOO rpm, wash with 70% Ethanol, dry and resuspend in 20ml sterile DEPC water.
Test 1ml on agarose gel. (generaly 1ml will be used for the hybridization).
In situ hybridization
- Fix embryos in 4% paraformaldehyde (PFA) in PBS overnight at 4°C.
- Remove chorions manually.
- transfer embryos in 100% Methanol (MeOH), store them at -20°C (2h-several months).
In situ Day 1 :
- Rehydratation :
put embryos is small baskets and rehydrate by successive incubtions in :
5mn in 75%MeOH-25%PBS
5mn in 50%MeOH-50%PBS
5mn in 25%MeOH-75%PBS
4 x 5mn in 100% PBT (PBS/tween20 0.1%)
- Digestionwith Proteinase K (10mg/ml)
Blastula and gastrula : Ø, early somitogenesis : 1mn; late somitogenesis (14 to 22 somites) : 4mn, 24h : 10mn, 36h/48h embryos : 20mn.
(One batch of embryos is used for the antibody preabsorption.
- Refixation 20mn in 4%PFA-PBS
- Washes 5 x 5mn in PBT
- Preadsorbtion of the anti-DIG antibody (Boehringer) in a 1000 dilution in PBT-sheep serum 2%-BSA 2mg/ml for several hours at room temperature with the batch of embryos previously prepared.
Prehybridization and hybridization :
tRNA and Heparin are added for prehybridation and hybridization only. (% of formamide varies function of the stringency).
Hybridization mix :
- Formamide 50 - 65%
- SSC 5 x SSC
- Heparin 50 µg/ml
- tRNA 500 µg/ml
- tween 20 0.1%
- citric acid to pH 6.0 (460 µl of 1M for 100 ml)
2 to 5 hrs at 70°C in 800 µl of hybridization mix.
Prehybridization mix is removed, discarded and replaced by 200 µl of hybridization mix containing 100 - 200 ng of antisens RNA probe. Hybridization is performed overnight, in a waterbath at 70°C.
Day 2 :
Washes : .
Embryos are transfered into small baskets fixed in floaters. Subsequent washes and incubation are carried out by letting the floater swim under agitation in 200 ml of the adequate solution.
- 1 x fast in 100 HM at 70°C.
- 15 mn in 75% HM/25% 2 x SSC at 70°C
- 15 mn in 50% HM/50% 2 x SSC at 70°C
- 15 mn in 25% HM/75% 2 x SSC at 70°C
- 15 mn in 2 x SSC at 70°C
- 2 x 30 mn in 0.2 x SSC (for normal stringency) or 0.05 x SSC (for high stringency).
- 10 mn in 75% 0.2 (0.05) x SSC/25% PBT at room temperature (RT)
- 10 mn in 50% 0.2 (0.05) x SSC/50% PBT at RT
- 10 mn in 25% 0.2 (0.05) x SSC/75% PBT at RT
- 10 mn in PBT at RT
- several hours in PBT/2% sheep serum/2mg:ml BSA at RT
Incubation with anti-DIG antiserum :
- Preadsorbed anti-DIG is used at the 5000 x dilution in PBT/2% sheep serum/2mg:ml BSA overnight with agitation at +4°C.
Antiserum is preadsorbed on the first day against
Day 3 :
Antiserum is removed and discarded. Embryos are then extensively washed :
- 1 x fast in PBT at RT
- 6 x 15 mn in PBT at RT
- 3 x 5 mn in solution buffer (100 mM tris HCl pH9.5, 50 mM MgCl2, 100 mM NaCl, 0.1% tween 20)
staining is performed at RT and monitored with a dissecting scope. Staining solution :
- 225 µl NBT 50 mg/ml
- 175 µl BCIP 50 mg/ml
- 50 ml staining buffer
(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 Dimethylformamide anhydre).
Reaction is stopped by removing the staining solution and washing embryos in stop solution : PBS pH5.5, EDTA 1mM. Embryos are then store in stop solution at +4°C in the dark.
- embryos can be observed directly in stop solution at the dissecting scope
- embryos can be mounted directly in 100% glycerol for observation at the compound microscope.
- Embryos at early developement stage (up to 18 hrs) are first dehydrated in 100% Methanol, then clarified few minutes in methylsalycilate and then mounted in Permount.
Materiels and supplies :
- paraformaldehyde (Sigma)
- 10 x PBS
- Tween 20 (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)
- desionized Formamide (high purity grade)
- 20 x SSC
- Heparin at 5 mg/ml (Sigma H3393)
- RNAse free tRNA (50 mg/ml) (Sigma R7876, resuspended in Water and extensively deproteinized by series of Phenol/CHCl3 extractions)
- 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)
- Waterbath at 70°C (with shaking).
- orbital shaker. (horizontal shaker).