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Revised Stern lab protocol for whole-mount in situ hybridization

Based mainly on the protocol by Domingos Henrique and David Ish-Horowicz, who in turn modified protocols from Ron Conlon, Richard Harland, Phil Ingham and David Wilkinson.

Transcription of DIG-riboprobe

Cut vector with appropriate enzyme, 4-5 hours or overnight.

After cutting, run agarose gel with 1/20 of cutting reaction.

Phenol:Chloroform extract in the fume hood (add an equal volume of Phenol [equil. pH 7.0-8.0*] : Chloroform 1:1, vortex hard, spin on microfuge for 1-2 min).

* DNA goes into the organic (phenol/chloroform) phase at acid pH but into the aqueous phase at alkaline pH.

Take top layer into clean Eppendorf and add 10µl 3M Na Acetate for every 100µl and 2.5x its volume of absolute alcohol. Vortex briefly and put at -20 oC. Minimum precipitation at this temperature or lower is about 1 hour. Otherwise leave overnight.

Spin 4 oC 15 min, take off alcohol. Probably won't see pellet but be careful not to touch it.

Wash with 150µl 70% EtOH, vortex, and spin again 5 min.

Take off alcohol carefully and dry pellet at 37 or 65 oC completely.

Add clean (preferably commercial Ultrapure, without traces of DEPC that may inhibit enzyme reactions) water to a final concentration of about 1mg/ml. Allow to dissolve at 37 or 65 oC for at least 15 min, with occasional vortexing or flicking the tube.

Transcribe with appropriate enzyme (T3, T7 or SP6). Proportions as follows (add in this order):

Transcription Mix

Transcribe at 37oC for 2-3 hours.

troubleshooting transcription with SP6: Use 2-3 times the amount of DNA template shown above for the transcription reaction, and transcribe at 40oC.

Add DNase (2 µl per µg DNA): first make up a stock of 2:10 DNase in transcription buffer with DTT (e.g.: 5 µl water, 1µl DTT, 2 µl transcription buffer, 2µl DNase I), then add this to tube. Incubate 30 minutes at 37oC.

At this point, take 1/20th of sample (2ul for small scale. 3ul for large scale) and run an agarose gel to check that the probe has been synthesized and that template DNA has been digested.

Make up volume to 80µl with water, and add 8µl EDTA to stop the DNAse.

Add 10µl 4M LiCl and 250µl absolute EtOH. Vortex briefly and put at -20 oC as above.

Precipitate overnight (min. 1 hour). Spin at 4 oC 15 min. Take off alcohol (this time you should see a good pellet).

Wash with 300 µl 70% EtOH. Vortex very well. The pellet should be dislodged and should break up into many little pieces, but be careful because it may stick to the upper part of the tube.

Spin again 5 min. Take off alcohol.

Wash again with 50µl absolute EtOH. Spin briefly and remove alcohol.

Allow to dry completely at 37-65 oC.

Dissolve pellet in 50µl water and leave at 37-65 oC for at least 15 min, vortexing occasionally.

Precipitate again by adding 10µl 4M LiCl and 250µl absolute EtOH, put at -20 oC overnight (min. 1 hour). Spin at 4 oC 15 min. Take off alcohol. Wash with 300 µl 70% EtOH. Vortex. Spin 5 min. Take off alcohol. Wash again with 50µl absolute EtOH. Spin briefly and remove alcohol and allow to dry completely at 37-65 oC. Dissolve pellet in water at 1mg/ml approx. (transcription should yield about 8x the original weight of the DNA). Leave at 37-65 oC for at least 15 min, vortexing occasionally.

Denature probe for 3 min at 95 oC and immediately cool on ice 5 min.

Spin briefly, then add 10x the volume of hybridization buffer for storage (or make up to about 100-200 ng/ml in hybridization buffer. Roughly 10-15ml for large scale transcription, 5-6ml for small scale transcription). Store at -20 oC.

For in situ hybridization with 2 probes: mix the two probes and perform the hybridization at the same time. Transcribe second probe exactly as above, except that instead of DIG-nucleotide mix one uses FITC-labelled UTP (Boehringer) mixed with unlabelled nucleotides to give the same proportions of nucleotides (see Boehringer sheet). Most important: for in situs with 2 probes, the complete removal of all unbound DIG and FITC is crucial for low background and good discrimination between the 2 probes. There are 2 main ways to achieve this: (a) pass the transcript, after dissolving it in water, through a RNase-free mini-G25 spin column; (b) precipitate each riboprobe twice.

Note: for in situ hybridization with 2 probes, it is important to use two strongly expressed genes. Label the strongest of these two with DIG and develop it second using INT-BCIP (brick red) or MagentaPhos-TetRed (magenta). Label the less strong one with FITC as above, and develop it first using NBT-BCIP.

Dissections

Collect embryos in calcium-magnesium-free (CMF) PBS, Pannett-Compton saline or Tyrodes saline.

Fix in freshly made 4% paraformaldehyde/CMF PBS/EGTA (4% w/v paraformaldehyde powder added to CMF PBS preheated at 65C. Adjust pH to about 7.5 with 1N NaOH. Allow to cool, then add EGTA to final concentration of 2mM.

Leave embryos for 1 hour at room temperature or overnight at 4C.

Note: prestreak embryos (very young embryos) are best fixed for 1 hour at room temperature, older embryos (HH5 and above) overnight at 4C. Probe specific optimization may be required.

 Transfer embryos to absolute methanol, and store in this for a minimum overnight and up to 1 week at -20oC. If embryos are to be kept longer before in situ, it is best to take them through to the first day pretreatments (until just before adding the probe) and store at -20oC in Hybridization solution (this seems to lead to no loss of signal, even if the embryos are kept virtually indefinitely in Pre-hyb).

First Day: Pretreatments

IMPORTANT NOTE ON WASHES: Take off and replace all solutions with a Pasteur pipette, not a Gilson. Tilt the vial and turn around to remove solutions with the pipette, without sucking the embryos into it. At each step, remove all the solution from vial until embryos stay in position attached to wall, but then add new fluid quickly so they don't dry. When adding new liquid, turn the vial so as to add it to the opposite side of the vial to where the embryos are, and let it run down the wall of the vial. We use 5 ml glass scintillation vials with screw-cap for the whole protocol.

For embryos older than about 2 days bleaching is required. Bleach either prior to rehydration in 6% H2O2 in 100% methanol for 1hr @ RT or after rehydration for 1 hour in 6% H2O2 in PTW for 1hr @ RT (1ml 30% H2O2 + 4ml)

Rehydrate embryos through 75%, 50% and 25% methanol in PTW allowing embryos to settle between changes. PTW=CMF PBS with 0.1% Tween-20. Make fresh MeOH dilutions and allow to cool before use.

Rinse 2x with PTW (PTW=CMF PBS with 0.1% Tween-20).

Add Proteinase K (1:1000; final conc. = 10µg/ml) diluted in PTW. Incubate at room temp for an amount of time to be determined by end user. Guidelines are ~1 minute per HH stage (e.g 5 minutes for HH5). Time may need to be increased for large embryos. Time may need to be reduced for New cultured embryos or very fragile embryos. Strength of Proteinase K is also batch dependant. During incubation, gently roll the tube every few minutes to make sure the sides and top of vial get wet with Proteinase K.

Take off Proteinase K and rinse carefully x2 with PTW.

Replace PTW with 4% paraformaldehyde in PTW (made as above but doesn't need to be fresh), containing 0.1% glutaraldehyde. Postfix 20-30 minutes at RT.

Prehybridization and hybridization

Note: some probes may benefit from being 'cleaned-up' by going through hybridization on spare embryos.

Remove postfixing solution and rinse 3x with PTW.

Remove PTW, and replace with 1 ml hybridization solution. Allow embryos to equilibrate (sink).

Replace with 1-2 ml hybridization solution (5 ml if using large vials). Embryos can now be stored at -20 oC or proceed with pre-hybridization at 70 oC.

Place tube upright in a beaker in water bath at 70 oC. Incubate 2-6 hours.

Remove hybridization solution, and replace with probe diluted in hybridization solution (see above "Transcription").

For probes <~400 nucleotides, it may be necessary to use a lower hybridization temperature (e.g. 62-65C for 100-200 nt, 65-68C for 200-350 nt.). Prehybridize at 70C, then lower the temperature when adding the short probe.

For in situ hybridization with 2 probes, add both probes simultaneously.

Hybridization solution

Second Day: Post-hybridization washes

Remove probe (keep for recycling at least 15 times it gets better!).

Rinse 3x with a small volume (<1ml) prewarmed hybridization solution.

Wash 2x with 1.5 ml (4 ml if large vial) prewarmed hybridization solution, 30 min in water bath.

Wash 20 min with prewarmed 1:1 hybridization solution : TBST (1:10 from the following stock)

10x TBST

20x TBS

Rinse 3x with TBST at room temperature

Wash 3x 30 min with TBST at room temperature

Incubate embryos in 1ml blocking buffer: 5% heat inactivated (at 55 oC for 30 min) goat serum + 1 mg/ml BSA in TBST, 3 hours at RT.

Dilute the anti -Digoxigenin AP antibody 1:5,000 in blocking buffer + 0.02% thimerosal. Antibody blocking buffer may also be made up using embryo powder (see recipe below).

Remove blocking buffer from embryos and replace with antibody. Incubate overnight at 4 oC on a rocking platform.

Note: when antibody is freshly made, it can be 'cleaned-up' by prior incubation on spare embryos.

Third Day: Post-antibody washes

Remove antibody solution (keep at 4 oC for recycling at least 15 times, it gets better with repeated use!).

Rinse 3x with TBST at RT

Wash 3x 1 hour with TBST, rocking (fill vial right up to the top) at RT. Older embryos need more washing, and embryos can be washed overnight or over the weekend at 4oC. If washing overnight or over the weekend at 4oC, rinse once more with room temperature TBST before proceeding with the next step.

Wash 2x 10 min with NTMT

NTMT

Incubate in NTMT containing 4.5 µl NBT (75mg/ml in 70% DMF) and 3.5 µl BCIP (50mg/ml in 100% DMF) per 1.5 ml, rocking, protected from light, at RT.

No need to look for the first 15 min. Thereafter look occasionally. Colour may take anything from 15 min to 3 days (or longer!) to develop at room temperature. If you want to go home and colour hasn't quite developed on the first day, leave the vials in the cold room overnight and take them out to room temp again the next day if necessary. At the end of the 2nd day there is no point in putting them in the cold room again.

After colour has developed as desired, stop by rinsing 3x in TBST or PBS/PTW (If using PBS or PTW ensure all NTMT is rinsed off). .

Finish by fixing in 4% paraformaldehyde and store in this if desired.

Fourth Day: Antibody washes and detection of 2nd probe for in situ hybridization with 2 probes, proceed from the last step as follows:

Fix at least overnight or 24 hours (or longer) in 4% paraformaldehyde (no glutaraldehyde) at 4 oC (to inactivate alkaline phosphatase).

Rinse 3x and wash 5x 30min (or more) in TBST containing 0.1% Tween (i.e rather than 1%). It is very important to remove all traces of paraformaldehyde as well as all traces of NBT-BCIP before the next step.

Further inactivate first alkaline phosphatase by incubating 45 min - 1 h in TBST/0.1% Tween at 65-70 oC.

Wash 3x 15min in TBST/0.1% Tween, and then 3x 15min in TBST/1% Tween (to remove any further traces of NBT-BCIP which are leached out in the cooking above).

Block as described above, 2 h in TBST/BSA/goat serum at RT.

Add anti-FITC antibody coupled to alkaline phosphatase in blocking buffer at a final concentration of 1:5,000. Incubate overnight at 4 oC.

TBST washes as described above.

NTMT equilibration, then incubate in 1.5 ml NTMT containing 7.5ul NBT/BCIP, rocking, protected from light, at RT.

OR:

NTMT containing 5 µl Magenta Phos (Molecular Probes; 50mg/ml in 100% DMF) and 4 µl Tetrazolium Red (Sigma, 75mg/ml in 70% DMF) per ml, rocking, protected from light, at RT.

Continue as described above for first probe (day 3). NB: Colour development with INT-BCIP is about the same as with NBT/BCIP (but after 3-4 hours background may start to come up), but with Magenta Phos/Tet Red can take considerably longer, often overnight or several days.

For 1- or 2-probe in situs, finish by fixing in 4% paraformaldehyde and store in this if desired.

For histological wax sectioning:

 Wash fixed embryos with PBS/PTW and dehydrate/clear as follows:

(a) absolute methanol, 10 min.

(b) propan-2-ol, 5 min.

(c) tetrahydronaphthalene, 30 min.

(d) 1:1 tetrahydronaphthalene:wax 60 oC, 30 min to 1 hour.

(e) 3x wax at 60 oC, 30 min.

(f) pour into moulds, allow to set overnight, then section as usual.

Subsequent dewaxing can be done in Histoclear.

Important note for 2-colour in situs: the reaction product of INT-BCIP does not survive dehydration in alcohols or clearing. For these it is necessary to cut frozen sections in a cryostat or a vibratome. MagentaPhos is OK.

Embryo Powder recipe:

(a) weigh X mg of embryo powder, where X=2x number of mls of final vol. of Ab. solution needed into an Eppendorf.

(b) add 500 µl TBST and vortex for 20 seconds

(c) heat to 70 oC for 30 minutes, vortex again 20 sec. and spin down at LOW speed for 1 min - just enough to get the powder to form a loose pellet.

(d) remove the supernatant and discard.

(e) wash the pellet x5 with 500 µl TBST, spinning low speed each time (this is to remove any fat that may be floating on the supernatant, which both makes a mess later and increases background for some reason). Repeat until no more fat at top of sup't.

(f) resuspend the pellet in 100 µl BLOCKING BUFFER (not TBST) for every bottle of embryos, mixing gently (not shaking). Incubate 10 min. RT. on rocker.

(g) add antibody so that the final concentration will be 1:5,000. For example: if your final incubation in antibody overnight will have 1 ml per tube, your embryos are now sitting in 1ml blocking buffer. If you have 5 bottles, add 1 µl antibody to the 500 µl blocking buffer with the embryo powder.

(h) After absorbing antibody with powder, spin down HARD (high speed) in the microfuge for 3 min. Keep the supernatant.