Wholemount in situ hybridisation

 

Wholemount in situ hybridisation

Based on Wilkinson protocol, modified by Murray Hargrave (m.hargrave@cmcb.uq.edu.au), Koopman lab

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A. SYNTHESIS OF PROBE

 

1 Mix in the following order at room temperature:

 

• sterile distilled water 9.5µL
• 5x transcription buffer 4µL
• 0.2M DTT 1µL
• nucleotide mix (pH 8.0) 2µL
• (10x DIG RNA labelling mix)
• linearised plasmid (1µg/µL) 1µL
• placental ribonuclease inhibitor (40U/µL) 1.5µL
• Sp6, T7 or T3 RNA polymerase (20U/µL) 1µL

 

2 Incubate at 37°C for 1 hour, then add another 20U of RNA polymerase.

 

3 Incubate for a further hour at 37°C.

4 Remove a 1µL aliquot and run on a 1% agarose/TAE gel to estimate the amount synthesised. An RNA band of approximately 10 fold greater intensity than the plasmid band indicates that ~10µg of probe has been synthesised.

 

5 There are two successful methods for purification of the RNA probe:

A) for the lazy (this works fine):

• Dilute the probe to 50µl with DEPC milliQ H2O, add 5µl of RNase free 3M NaOAc, mix and add 2.5 volumes of RNase free abs. ethanol.
• Incubate at -20°C for 30 minutes to preciptate the RNA and spin down in a refrigerated microfuge for 10 minutes.
• Wash the pellet well (twice) with RNase free 70% ethanol to get rid of any unincorporated nucleotides

 

B) for the paranoid:

• Add 2µL of RNase free DNase1 (10U/?l). Incubate at 37°C for 15 minutes.
• Pack a Sephadex G50 column equilibrated with 0.1% SDS, 50mM Tris.Cl (pH 7.5), 0.5mM EDTA (TES) by loading a 1ml syringe barrel (plugged with glass or plastic wool) and spinning @ 1500rpm 90sec.
• Dilute probe reaction to 100?l with TES and load onto packed G50 column. Collect fraction and load another 100?l to the column and spin again.
• Pool the fractions and add 1/10 volume NaOAc, 2 volumes 100% EtOH and incubate at -20°C for 30 minutes.
• Spin in a refrigerated microfuge for 10 minutes, wash pellet with ice-cold 70% EtOH
• Air dry pellet

 

6 Redissolve pellet in DEPC milliQ H2O at ~0.1µg/µL and store at -20°C.

 

 

B. PRETREATMENT OF EMBRYOS

• All steps are carried out in 4mL round-bottom polycarbonate snap-cap tubes unless otherwise stated. Enough liquid must be used to ensure that all embryos are completely covered during each step.
• All steps are carried out with sufficient rocking to agitate (but not destroy) the embryos, and unless otherwise stated, at room temperature.
• All steps up to and including hybridisation are carried out in RNase-free conditions, using RNase-free solutions, wearing gloves etc.
• For the washes at 55°C or 65°C, it is convenient to use a heater block placed on its side on a rocking platform or a hybridization oven with rotating cylinders.

 

 

Day 0

 

1 Dissect embryos in ice-cold PBS. Try to remove as much of the extraembryonic membranes as possible. Be sure to remove or at least puncture the amnion and in embryos of 10dpc or older, puncture the head with a syringe needle to avoid trapping of reagents in the lumen.

 

2 Fix in 4% paraformaldehyde (PFA) in PBS at 4°C overnight.

Varying the fixation time from 3h to overnight has no effect on signal or background.

 

3 Wash twice with PBTX for 10 minutes each at 4°C.

 

4 Wash with 50%, MeOH/PBTX, then twice with 100% MeOH for 10 minutes each.

Can store the embyros at 4°C or -20°C at this point, for up to a few months, or preferably in pre-hyb (see step 12).

 

Day 1

 

5 Rehydrate by taking the embryos back through a MeOH/PBTX (75%MeOH -> 50% MeOH -> 25% MeOH -> PBTX) series in reverse

 

6 Wash twice with PBTX for 10 minutes each.

 

7 Treat with 10µg/mL ProteinaseK in PBTX for 5-20 minutes at room temperature.

The length of this treatment depends on the size of the sample and the batch of proteinase K. Each batch should ideally be tested. As a rough guide, use 5 min for E7.5, 7 min for E8.5, 9 min for E9.5, 11 min for E10.5, 12-14 min for E11.5.

 

8 Wash twice with PBTX for 5 minutes each.

Be careful - the embryos are fragile!

9 Refix with fresh 0.2% glutaraldehyde/4% PFA in PBTX for 20 minutes.

10 Wash twice with PBTX for 10 minutes each.

 

11 Place in a 2ml screw cap eppendorf tube and fill with prehybridisation mix, and allow the embryos to sink, replace prehyb soln.

 

12 Incubate at 65°C for 2h.

This step can also be performed overnight. Alternatively the embryos can be stored in this solution at -20°C.

13 Remove prehyb and add hybridisation mix including 1.0 µg/mL DIG labelled RNA probe.

If high background is seen, probe concentration can be decreased to 0.5 µg/mL.

The tube needs to be full so that probe does not dry onto the sample.

14 Incubate at 65°C overnight.

If the probe is short or heterologous, 55°C can be used for pre-hyb, hyb and stringency washes.

 

Day 2

 

C. POST-HYBRIDISATION WASHES

From this point on RNase-free conditions are no longer necessary.

1 Wash with the following for 5 minutes each at 65°C (or 55°C)

• 100% Solution 1
• 75% Solution 1 : 25% 2xSSC
• 50% Solution 1 : 50% 2xSSC
• 25% Solution 1 : 75% 2xSSC

During these washes, start preabsorbing the antibody as described below.

2 Wash with 2xSSC, 0.1% CHAPS twice for 30 minutes each at 65°C (or 55°C).

3 Wash with 0.2xSSC, 0.1% CHAPS twice for 30 minutes each at 65°C (or 55°C).

4 Wash with TBTX, twice for 10 minutes each at room temperature.

5 Preblock the embryos with 10% sheep serum, 2% BSA in TBTX for 2-3 hours at room temperature.

6 Remove the 10% sheep serum, 2% BSA from the embryos and replace with the preabsorbed antibody (see below). Rock overnight at 4°C.

 

 

D. PREABSORPTION OF ANTIBODY

 

1 During the washing of the embryos (step 1 above), weigh out 3mg of embryo powder into a microtube, add 0.5mL of 10% sheep serum, 2% BSA in TBTX and 1µL anti-DIG-AP Fab fragment (Boehringer 1093274).

Embryo powder should match the species being studied.

2 Rock gently at 4°C for 3 hours or longer.

3 Spin in a microfuge for 10 minutes at 4°C.

4 Dilute the supernatant to 2mL using 10% sheep serum, 2% BSA in TBTX.

5 Store at 4°C until use.

 

E. POST-ANTIBODY WASHES AND HISTOCHEMISTRY

 

Day 3

1 Wash at least five times with TBTX containing 0.1% BSA for 1 hour each at room temp.

The antibody solution can be kept at 4°C and reused up to 4 times.

2 Wash overnight at 4°C with TBTX containing 0.1% BSA.

This wash is optional but usually convenient.

 

 

Day 4

 

3 Wash twice with TBTX for 15 minutes each.

4 Wash three times with NTMT for 10 minutes each.

 

5 Incubate with NTMT including 4.5µL NBT and 3.5µL BCIP (X-phosphate) per mL. Rock for the first 20 minutes then transfer the embryos to a glass embryo dish or scintillation vial.

Avoid using a plastic petri dish as crystals tend to form.

 

Keep in the dark as much as possible and allow the colour reaction to proceed until signal is strongest without producing background staining.

It is best to slightly overstain, as subsequent washings will tend to destain samples. If samples are to be sectioned, overstaining is recommended. You can stop the colour reaction by washing in NTMT, then TBTX overnight, then re-start the colour reaction the next morning.

 

6 When the colour has developed to the desired extent, wash with NTMT then with PBTX.

 

7 Wash several times in PBS with 1% Triton X-100.

This will blue the stain and decrease background and signal. Some observation and judgement is required here. For weak signal, this step can be shortened or omitted. If signal is strong and background is weak, then a total of a few hours is recommended. Overstained or high background samples can be washed for up to several days.

 

8 Fix the stain by incubating the embryos in 4% PFA in PBTX overnight at 4°C.

 

9 Photograph embryos as soon as possible.

The signal can fade or the enitre embryo can turn blue upon storage. Position embryos, immersed in PBS, in grooves cut in a layer of agarose in a petri dish. Adjust lighting to optimize the translucency of the sample.

 

10 If the embryos are to be stored for extended periods, use PBS containing sodium azide, or take them through a PBTX/glycerol series into 100% glycerol.

 

 

SOLUTIONS

 

• 10x transcription buffer (for probe preparation): 400mM Tris.Cl (pH 8.25), 60mM MgCl2, 20mM spermidine. This is supplied with Promega polymerases.
• Nucleotide mix : 10mM GTP, 10mM ATP, 10mM CTP, 6.5mM UTP, 3.5mM DIG-UTP. This can be bought from Boehringer Mannheim.
• 4% PFA in PBS (pH 7.4) - prepared ahead of time by dissolving the powder at 65°C, and then cooled. This can be frozen in aliquots.
• 25% glutaraldehyde : store in aliquot amounts at -20°C and thaw just prior to use.
• PBTX : PBS (pH 7.4) with 0.1% Triton X-100
• Prehybridisation mix : 50% formamide, 5xSSC, 2% Boehringer blocking powder (cat. no. 1096176, dissolve directly into the mix), 0.1% Triton X-100, 0.5% CHAPS (Sigma C-3023), 50 µg/mL yeast RNA (Sigma R-6625), 5mM EDTA, 50µg/mL heparin. For hybridisation, add probe to 1 µg/mL
• Solution 1 : 50% formamide, 5xSSC, 0.1% Triton X-100, 0.5% CHAPS
• TBTX : 50mM Tris.Cl (pH 7.5), 150mM NaCl, 0.1% Triton X-100
• Mouse embryo powder : Homogenise ~12.5-14.5dpc mouse embryos in a minimum volume of PBS. Add 4 volumes of ice-cold acetone, mix and incubate on ice for 30 minutes. Spin at 10,000x g for 10 minutes and remove supernatant. Wash the pellet with ice-cold acetone and spin again. Spread the pellet out and grind into a fine powder on a sheet of filter paper and allow it to air dry. Store in an air-tight tube at 4°C.
• NTMT : 100mM NaCl, 100mM Tris.Cl (pH 9.5), 50mM MgCl2, 0.1% Tween-20.

This must be made fresh on the day of use as pH decreases during storage due to the absorption of CO2.

NBT : 75mg/mL in dimethylformamide (store at -20°C)
• BCIP : 50 mg/mL in dimethylformamide (store at -20°C)

It is best to let these reagents warm to room temperature before use as it may decrease the amount of crystal formation in the colour reaction.

 

 

REFERENCE

 

Wilkinson, D.G. (1992) Whole mount in situ hybridization of vertebrate embryos, in In situ hybridization: A Practical Approach (D.G. Wilkinson, ed) pp 75-83, IRL Press, Oxford.