Whole-mount in situ hybridization

(Ch. Thisse and B. Thisse, IGBMC, 1 rue Laurent Fries, 67404 ILLKIRCH Cedex, FRANCE)

 

            Probe :

 

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 37C

- Digest the template DNA by adding 2ml RNAse free DNAse during 15mn at 37C.

- 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-70C during 30mn, centrifuge at 4C, 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 4C.

- Remove chorions manually.

- transfer embryos in 100% Methanol (MeOH), store them at -20C (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 : , 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 g/ml

            - tRNA                         500 g/ml

            - tween                        20        0.1%

            - citric acid     to pH 6.0 (460 l of 1M for 100 ml)

 

            Prehybridization :

2 to 5 hrs at 70C in 800 l of hybridization mix.

            Hybridization :

Prehybridization mix is removed, discarded and replaced by 200 l of hybridization mix containing 100 - 200 ng of antisens RNA probe. Hybridization is performed overnight, in a waterbath at 70C.

 

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 70C.

            - 15 mn in 75% HM/25% 2 x SSC at 70C

            - 15 mn in 50% HM/50% 2 x SSC at 70C

            - 15 mn in 25% HM/75% 2 x SSC at 70C

            - 15 mn in 2 x SSC at 70C

            - 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 +4C.

            Antiserum is preadsorbed on the first day against

 

Day 3 :

 

            Washes :

            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 :

 

            staining is performed at RT and monitored with a dissecting scope. Staining solution :

 

                        - 225 l NBT 50 mg/ml

                        - 175 l 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 +4C in the dark.

 

            Mounting :

           

            - 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.

 

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Materiels and supplies :

 

            - paraformaldehyde (Sigma)

            - 10 x PBS

            - Methanol

            - 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 70C (with shaking).

            - orbital shaker. (horizontal shaker).