WHOLE MOUNT IN SITU HYBRIDIZATION.Mouse Embryos.
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June, 1997 - optimized by N. Kertesz, L.Leyns & E. De Robertis
Please cite: Belo J.A, Bouwmeester T., Leyns L.,Kertesz N., Gallo M., Follettie M. and De Robertis E.M. "Cerberus-like is a secreted factor with neuralizing activity expressed in the anterior primitive endoderm of the mouse gastrula" Mechanism of Development, in press - 1997.
Modified from the protocol of Harland (1991) and Current Protocols in Molecular Biology.
- Probes are prepared as Digoxigenin labelled RNA . The labelling mix as well as all antibodies are purchased from Boehringer
- All conditions and solutions should be totally RNAse free.
- Use gloves and aerosol barrier tips.
- Linearize the plasmid and check the digest.
- Phenol extract.
- Extract twice with chloroform:isoamyl alcohol (24:1)
- Ethanol precipitate ( 1/2 vol 7.5 M NH4 OAc + 2.5 vol 100% ethanol. Rinse with 125µl 75% ethanol. Let dry with caps open for 10 minutes. )
- Resuspend in suitable volume of nuclease free water.
- Measure concentration.
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Set up transcription reaction
- 200ng DNA
- 2 µl 10X transcription buffer (Stratagene)
- 2 µl labelling mix
- 1 µl RNAGUARD (Pharmacia)
- 1 µl RNA polymerase (SP6, T3 or T7)
- Add water to 20 µl
- Incubate for 2 hours at 37 °C.
- Run on 1% agarose gel (1.5µl probe + 5µl of 1.2X running buffer.) for a short time. The RNA should appear as a single band with little degradation product and about 10 times more intense than the DNA band.
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Remove unincorporated free nucleotides with Quick-Spin Columns.
- Remove the caps (top first not to create air bubble trapped in the column) and spin for 5 min, @ 4 °C, 1800 rpm in the Sorval swing bucket centrifuge.
- Remove the eluate and centrifuge 5 min again.
- Put the column in new tubes, add the transcription reaction onto them and spin 15 min.
- The volume of the final eluate should be around 30-40µl .
- Run a gel (loading 1.5µl in 5µl loading buffer) to quantify the yield and to determine the amount to be used for the in situ .
- Don't let the embryos dry at any stage as the amount of background will increase. It is prefered to leave the embryos in a small volume of the solution and to add the next solution to it.
- Treat all solutions with DEPC (add 0.1% DEPC, incubate with agitation overnight and autoclave 40 minutes) (for Tris solution, use DEPC treated water, do not treat the solution).
- Filter all solutions (to remove particles that will stick to the embryos).
- Rinse the hybridization vials and caps with RNAZap and rinse at least 5 times with DEPC water.
- Make all the fixations, rinses, washes until the pre-hybridization step on ice except the proteinaseK treatment.
- Use gloves and aerosol barrier tips for changing the solutions from the fixation step to the end of hybridization.
- Dissect embryos in cold PBS , change solution often.
- Punch a hole in brain cavities for embryos older than 9 dpc.
- Transfer after dissecting a few embryos to a 5 ml screw cap flat bottomed glass vial containing 4% paraformaldehyde (freshly made. Add powder paraformaldehyde to PBS and heat to 60 °C with stirring until clear) Store on ice.
- When all the embryos of the same mother are dissected, renew the 4% paraformaldehyde and incubate at 4 °C for 4 hrs for 7.5d embryos or overnight for older embryos (or overday if dissection is done in the morning).
- The next day, wash 2x with PBSw ( PBSw=PBS with 0.1% Tween-20)
- Dehydrate with methanol series (25%, 50%, 75%, 100% in PBSw). Change 2x in 100% methanol.
- Store the embryos at -20 °C (up to 2 months).
In Situ hybridization
- Prepare fresh 4% paraformaldehyde - 0.2% Glutaraldehyde in PBS. About 5 ml will be needed for each sample after proteinaseK treatment.
- Prepare hybridization solution. For 50 ml of hybridization solution dissolve;
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Hybridization mix recipe (50 ml):
- 0.5 g Boehringer Block
- 25 ml formamide
- 12.5 ml 20X SSC, pH 7
- Heat to 65 °C for about 1 hr. Once dissolved add:
- 6 ml H2O
- 5ml 10mg/ml torula RNA(heat 2 min at 65 C to clear)
- 100 µl 50mg/ml heparin
- 250 µl 20% Tween-20
- 500 µl 10% CHAPS
- 500 µl 0.5 M EDTA
- Filter the solution. The hybridization solution can be prepared before, aliquoted and stored at -20 °C.
- Rehydrate the embryos through 75, 50 and 25% methanol series in PBSw. Incubate each step for 5 min. on ice.
- Wash 3 times for 5 min. with PBSw on ice.
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Change to 1ml of 4.5 µg/ml Proteinase K in PBSw.
Incubate for 3 min for 6dpc at RT, 5 min for 7.5dpc, 7 min for 8.5 dpc, 9 min for 9.5 dpc, 11 min for 10.5 dpc, 13 min for 11.5 dpc.
Staining for highly expressed gene requires less digestion, but for low expression genes longer digestion may help to get stronger staining. Make sure to thaw the proteinase K stock completely and vortex to dissolve precipitate at the bottom of the tube.
Use aliquots of the proteinase K stock 10mg/ml, do not thaw-freeze repeatedly. (Incubation times have to be optimized for each stock.) - Stop digestion by washing in freshly prepared 2mg/ml glycine in PBSW
- Rinse in PBSw.
- Wash 2 times with PBSw for 5 min .
- Refix in 5 ml of 4% paraformaldehyde-0.2% glutaraldehyde in PBSw for 15 min.
- Rinse in PBSw.
- Wash 3 times with PBSw for 5 min. each.
- Wash in 1 ml of 50% PBSw:50% hybridization solution, followed by 100% hybridization solution for about 3 min. each standing.
- Replace 900 µl of fresh hybridization mix in each glass vial.
- Prehybridize samples for 3 hrs at 65 °C.
- Heat 200 ng of the RNA probe in 100 µl of hybridization mix to 95 °C for 5 min.
- Add the probe/hybridization mix to the embryos. The final probe concentration should be about 200ng/ml.
- Hybridize overnight at 70 °C in a water bath.
In Situ hybridization
- Remove hybridization solution and add 800µl of prehybridization solution. Wash for 5 minutes at 70 °C.
- Add 400µl of 2X SSC, ph 4.5 (without removing prehybridization solution.) C. Repeat the addition of the 2XSSC wash twice more.
- Remove the mix and wash twice, 30 min each time, in 2XSSC pH7/0.1% CHAPS 70 °C.
- Wash twice, 10 min each, in Maleic Acid Buffer ( MAB; 100 mM maleic acid, 150 mM NaCl; pH 7.5) at room temperature. Wash twice, 30 min each time, in MAB at 70 °C.
- Wash twice 10 min each in PBS at room temperature.
- Wash 5 min in PBSw at room temperature.
- Incubate the embryos in 1 ml antibody buffer for at least 2 hours at 4 °C with rocking.
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BMblock-mouse antibody buffer 2.5ml needed for each sample:
- 10% Goat serum (heat inactivated 30 min at 56 °C)
- 1% boehringer blocking reagent in PBSw
- Heat the mixture at 65 °C until total dissolution, filter through 4.5 micron filters (several may be needed), then cool on ice.
- During the blocking step, preabsorbe the antibodies. The dilution for the Alkaline phosphatase conjugate (AP) is 1/10000 from a stock of 150 units/200 µl (Boehringer). Dilute the antibody in 1.5 ml of antibody buffer and incubate rocking for at least 2 hours at 4 °C. Use this solution to replace the blocking solution.
- Replace buffer with diluted antibody and incubate overnight at 4 °C rocking.
In Situ hybridization
- Fast wash embryos with 0.1% BSA in PBSw.
- Do another 5 washes with 5 mls 0.1%BSA in PBSw (fill to the top to minimize air bubble) rotating for 45 min. each.
- Wash twice, 30 min. each in PBSw.
- Take out staining solutions to warm in RT.
- Wash the embryos in AP1 buffer (100 mM Tris 9.5; 100 mM NaCl; 50 mM MgCl2) rocking for 10 min, two times each, at RT.
- Replace with 1 ml BM purple and rock slowly in the dark. BM purple staining takes a few hours to several days, if necessary leave at 4 °C overnight or until background appears.
- Stop staining reaction by washing in at least three changes of PBS.
- After staining, dehydrate through methanol series (25%, 50%, 75%, 2x 100%) and store in methanol at -20 °C.
- Take pictures after placing back in methanol. BM purple becomes more blue and intense in methanol.
