WHOLE-MOUNT IN SITU HYBRIDIZATION ON VERTEBRATE EMBRYOS

Domingos Henrique and David Ish-Horowicz, Imperial Cancer Research Fund, P0 Box 123, 44 Lincolns Inn Fields, London WC2A 3PX. Fax: 44 171 3417. E-mail:
[email protected] uk or [email protected] uk).

This in situ recipe is working well in many labs. Originally developed for chick embryos, it also works well on mice, Xenopus and fish embryos. Please use and distribute it freely, citing Henrique et al. Nature 375: 787-790 (1995) for publication.

Modified from protocols of Ron Conlon (Mt. Sinai, Toronto), Phil Ingham (ICRF Oxford), David Wilkinson (NIMR, London) and Richard Harland (UC, Berkeley) with various other inputs. Note hybridization in much reduced salt concentration (i.e. at high stringency) and omission of RNAse digestion. This gives specificity, sensitivity and low background. The recipe has worked with a 264bp RNA probe.

Available by anonymous ftp at ftp.lif.icnet.uk/icrf-public/recipes in two alternative file formats: insiturecipe11_95.hqx (binhexed Macintosh Word v5.1 format); insiturecipe11_95.rtf (Microsoft interchange (RTF) format; ASCII text that can be imported/translated by most word processors).

DISSECTIONS
1.Dissect (chick) embryos in PBS + 2mM ECTA; remove as much of the extra- embryonic membranes as possible (except stages <8).

2.Fix in l0ml 4% formaldehyde (HCHO) in PBS + 2mM EGTA, 1-2h at room temp; or 4 0C, 2h-o/n. (Can use MEMFA buffer for Xenopus.)

3.Wash twice in PTW

4.Wash with 50% MeOH/PTW, then 100% MeOH twice; can store at this point at -20 oC (for less than one month).

PTW = PBS, 0.1% Tween-20.

PRETREATMENTS and HYBRIDIZATION

5.Rehydrate embryos through 75%, 50%, 25% MeOH/PTW (allowing embryos to settle), and washing twice with PTW
6.Treat with 10ug/ml proteinase K in PTW: For chick embryos, time of incubation in min A stage no. For mice: 6.5-7.5 days - 5min; thereafter 5min extra/day up to 10.5d. Xenopus: gastrulae - l0min; neurulae - 15min; >stage 25 - 30min.

7.Remove proteinase, rinse briefly (care !) with PTW, and post-fix for 20min in 4% HCHO +0.1% Glutaraldehyde, in PTW

8.Rinse and wash once with PTW. Transfer embryos to 2m1 round-bottom microtube.

9.Rinse once with 1:1 PTW/hybridization mix. Let embryos settle.

10.Rinse with 1 ml hybridization mix. Let embryos settle.

11.Replace with 1ml hybridization mix and incubate horizontally 1h/65 0C (for Xenopus, we used 60 0C here and in subsequent high temperature incubations).

[Can store at -200C before or after prehybridizing.]

12.Add lml pre-warmed hybridization mix @ 1ug/ml DIG-labelled RNA probe (possibly 0.1 ug/ml is enough). Immediately place at 65 0C.

13. Incubate o/n horizontally at 65 0C (60 0C for Xenopus) [Rock once after 20-30min].

Hybridization mix:
Final concn.
Formamide (Fluka 47670) 50% 25ml
SSC (20x pH5 w citric acid!!) 1.3xSSC 3.25ml
EDTA (0.5M, pH8) 5mM 0.5ml
Yeast RNA (2Omg/ml) (Sigma R-6625) 50ug/ml 125ul
Tween-20 (10%) 0.2% lml
CHAPS (10%) 0.5% 2.5ml
Heparin (5Omg/ml) (Sigma H-7005) 100mg/ml 100ul
H20 17.5m1
Total 50ml

°Steps 1-9 are carried out on a roller, in 3-5ml solution in 6ml round bottomed Falcon tubes. Thereafter, use 1.7-2m1 in a 2m1 microtube, and rocking at room temperature unless otherwise stated. In this recipe, rinses are immediate, and washes are for 5min unless otherwise stated.

°4% formaldehyde/PBS should be made on day of use by diluting 1vol 40% HCHO with 9vols PBS. Add NaOH 1M to pH 7.5.

°10% Tween (v/v) = llg/lOOml and should be stored frozen at -20 0C.

°Hybridization mix can be stored at -20 0C.

°A stock of 25% glutaraldehyde is stored in aliquots at -20 0C. Thaw out aliquot just before use.

POST-HYBRIDIZATION WASHES

1.Rinse twice with prewarmed (65 0C) hybridization mix.

2.Wash 2 x 30 min/65 0C with 1.5m1 prewarmed hyb mix.

3.Wash 10 min/65 0C with 1.5m1 prewarmed 1:1 hyb mix/MABT.

4.Rinse 2x with 1.5m1 MABT (=l00mM Maleic acid, l50mM NaCl, pH7,5, 0.1% Tween-20).

5.Wash 1 x 15min with 1.5m1 MABT

6.Incubate lh with 1.5m1 MABT +2% Boehringer Blocking Reagent (BBR).

7.Incubate 1h in 1.5m1 MABT + 2% BBR + 20% heat-treated serum

8.Incubate o/n at 4 0C (or 4h at RT) in (fresh) 1ml MABT + 2% BBR + 20% serum +

1/2000 dilution of AP-anti-DIG antibody (Boehringer).

5xMAB(T) = Maleic Acid (M-0375) 11.6g

NaCl 8.7g
Tween20 11g
H2O ~185ml
Total 200m1

[Weigh, dissolve and pH maleic acid before adding other ingredients].

Steps 2 and 3: incubate in oven with the tubes horizontal. Steps 5-8: roll at room temperature.

°After each 65 0C wash, let embryos settle by incubating tube vertically at 65 0C in a heating block, then change supernatants individually so samples don't cool. Keep wash solutions at 65 0C in water-bath.

°Sheep serum is heat-treated at 55-60 0C, 30 min and stored in quick-frozen aliquots at -20 0C. Thawed aliquots can be stored at 4 0C with addition of azide to 0.1%.

°Boehringer Blocking Reagent (#1096 176). Make 10% stocks in MAB (no Tween) heating to dissolve, then autoclave, aliquot and freeze. Works better than "embryo powder", and is easier and more reproducible to make. We have used MABT thereafter for consistency, but maleate does not buffer well, and alternative buffers probably substitute as well or better.

POST-ANTIBODY WASHES AND HISTOCHEMISTRY

1.Rinse 3 times with 1.5m1 MABT. Transfer to glass scintillation vial.

2.Wash 3xlh with 10-20m1 MABT, by rolling.

3.Wash 2x10 mm with 10-20m1 NTMT.

4.Incubate with 1.5m1 NTMT + 4.5~l/ml NBT (75mg/ml in Dimethyl Formamide)

+3.5ul/ml BCIP (X-phosphate;5Omg/ml in 70% DMF). Rock for first 20min, then incubate at room temp. for 30min to 3days. Optional: doubling the NBT and BCIP concentration speeds up staining and doubles the cost. (Optional: staining develops faster at 37 0C but monitor very carefully).

5.When colour has developed to the desired extent, rinse lx and wash 2x with PTW. [To clear mouse embryos, bleach for lh in 6% H202/PBS; wash well with PTW]. Refix in 4% HCHO/0.1% glutaraldehyde/PTW for 2h (at room temp) or o/n (at 4 0C). Rinse lx and wash 2x l0min with PTW. Store at 40C in PTW + 0.1% azide.

NTMT: 5M NaCl lml

2M TrisHCl pH9.5 2.5ml
2M MgCl2 1.25m1
10% Tween-20 5m1
H20 40.25m1
Total 50m1

Make from stocks on day of use. NB, final Tween concentration is 1%. [Can re-use antibody; store with 0.025% azide.]

°Final note: Though not necessary in our hands, reducing the salt during hybridization (to lxSSC) may improve background further. Washes at higher stringency are irrelevant because, once formed, mismatch RNA-RNA hybrids never melt.