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.

• Preparation of the probe.

• 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.

1. Linearize the plasmid and check the digest.
2. Phenol extract.
3. Extract twice with chloroform:isoamyl alcohol (24:1)
4. 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. )
5. Resuspend in suitable volume of nuclease free water.
6. Measure concentration.
7. 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
8. Incubate for 2 hours at 37 °C.
9. 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.
10. 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.

1. Dissect embryos in cold PBS , change solution often.
2. Punch a hole in brain cavities for embryos older than 9 dpc.
3. 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.
4. 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).
5. The next day, wash 2x with PBSw ( PBSw=PBS with 0.1% Tween-20)
6. Dehydrate with methanol series (25%, 50%, 75%, 100% in PBSw). Change 2x in 100% methanol.
7. Store the embryos at -20 °C (up to 2 months).

In Situ hybridization

1. Prepare fresh 4% paraformaldehyde - 0.2% Glutaraldehyde in PBS. About 5 ml will be needed for each sample after proteinaseK treatment.
2. Prepare hybridization solution. For 50 ml of hybridization solution dissolve;

• 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.

1. Rehydrate the embryos through 75, 50 and 25% methanol series in PBSw. Incubate each step for 5 min. on ice.
2. Wash 3 times for 5 min. with PBSw on ice.
3. 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.)
4. Stop digestion by washing in freshly prepared 2mg/ml glycine in PBSW
5. Rinse in PBSw.
6. Wash 2 times with PBSw for 5 min .
7. Refix in 5 ml of 4% paraformaldehyde-0.2% glutaraldehyde in PBSw for 15 min.
8. Rinse in PBSw.
9. Wash 3 times with PBSw for 5 min. each.
10. Wash in 1 ml of 50% PBSw:50% hybridization solution, followed by 100% hybridization solution for about 3 min. each standing.
11. Replace 900 µl of fresh hybridization mix in each glass vial.
12. Prehybridize samples for 3 hrs at 65 °C.
13. Heat 200 ng of the RNA probe in 100 µl of hybridization mix to 95 °C for 5 min.
14. Add the probe/hybridization mix to the embryos. The final probe concentration should be about 200ng/ml.
15. Hybridize overnight at 70 °C in a water bath.