WHOLE-MOUNT IN SITU HYBRIDIZATION ON CHICKEN EMBRYOS
(Chitnis et al., 1995; Henrique et al., 1995)
Domingos Henrique and David Ish-Horowicz. (ICRF Dev. Biol. Unit, Oxford; Fax 0865 281310)
Modified from protocols of Ron Conlon (Mt. Sinai, Toronto), Phil Ingham (ICRF Oxford) and David Wilkinson (NIMR, London). Note HYBRIDISATION in much reduced salt concentration and omission of RNase digestion. New conditions for antibody detection are derived from Harland's lab protocol for frogs. Also tried on mouse embryos (7.5-10.5 dpc), and Xenopus embryos, successfully.
Dissections
1. Dissect embryos out in PBS + 2 mM EGTA; remove as much of the extra-embryonic membranes as possible.
2. Fix in 10 ml 4% formaldehyde (HCHO) in PBS + 2 mM EGTA, 1-2h at room temp; or 4°C, 2h.
3. Wash three times in PTW (= PBS, 0.1% Tween-20) and once for 1 hour.
4. Transfer to 100% MeOH; can store at this point at -20°C (less than one month).
Pretreatments and hybridization
5. Rehydrate embryos through 75%, 50%, 25% MeOH/PTW (allowing embryos to settle), and washing three times with PTW.
6. Treat with 10 µg/ml proteinase K in PTW for 7 minutes at 37°C (prewarmed solutions!).
7. Remove proteinase, rinse twice briefly (carefully and quickly!) with PTW, and post-fix for 20 min in 4% HCHO + 0.1% Glutaraldehyde, in PTW.
8. Rinse and wash once with PTW.
9. Rinse once with 1:1 PTW/HYBRIDISATION mix. Let embryos settle.
10. Rinse with 1 ml hybridization mix. Let embryos settle.
11. Replace with 1 ml hybridization mix and incubate with gentle mixing ³ 1h @ 65°C.
[Can store at -20°C (before or) after prehybridizing.]
12. Add 1 ml pre-warmed hybridization mix @ ~1 µg/ml DIG-labeled RNA probe (possibly 0.1 µg/ml is enough)
13. Incubate with gentle mixing at 65°C/overnight.
•Steps 1-4 are carried out in 15 ml falcon tubes, subsequent steps in 1.7-2 ml in a 2 ml microtube rocking at room temperature unless otherwise stated. Unless otherwise stated, rinses are immediate, and washes are for 5 min.
•A stock of 8% glutaraldehyde is stored in aliquots at -20
°C. Thaw out aliquot just before use.Hybridization mix
:|
Formamide |
50% |
25 ml |
|
SSC (20x pH 5 w citric acid!!) |
1.3 x SSC |
3.25 ml |
|
EDTA (0.5M, pH 8) |
5 mM |
0.5 ml |
|
Yeast RNA (20 mg/ml) |
50 µg/ml |
125 µl |
|
Tween-20 (10%) |
0.2% |
1 ml |
|
CHAPS (10%) |
0.5% |
2.5 ml |
|
Heparin (50 mg/ml) |
100 µg/ml |
100 µl |
|
H2O |
17.5 ml |
|
|
Total |
50 ml |
Post-hybridization washes
1. Rinse twice with prewarmed (65°C) hybridization mix.
2. Wash 10 min/65°C with prewarmed hyb mix.
3. Wash 2x30 min/65°C with Washing solution 1 (50% Formamide/1 x SSC/ 0.1% Tween-20), prewarmed at 65°C.
4. Wash 10 min/65°C with prewarmed 1:1 Washing solution 1/Maleic Acid Buffer (MABT: 100 mM maleic acid [Sigma M0375], 150 mM NaCl, 0.1% Tween-20, final pH 7.5).
5. Rinse 3 times with MABT.
6. Wash 2 x 30 min with MABT.
7. Replace with MABT + 2% BBR (Boehringer Blocking Reagent [BM 1096 176], make 10% stock in MAB by heating to dissolve, autoclave, aliquot and freeze).
Wash for 1 hour at room temp.
8. Preincubate in 2 ml of MABT+ 2% BBR + 20% heat treated goat serum (650C for 30 min), for 1-2 hours.
9. Replace with a solution of MABT + 2% BBR + 20% serum, containing a 1/5000 dilution of anti-DIG-AP antibody (BM 1093 274). Incubate overnight at +40C.
• After each 70°C wash, let embryos settle by incubating tube vertically at 70°C, then change supernatants individually so samples don't cool. Keep wash solutions at 70°C in water-bath.
• Serum is heat-treated at 65
°C, 30 min and stored in quick-frozen aliquots at —20°C. Thawed aliquots can be stored at 4°C with addition of azide to 0.1%.• If using a probe labeled with UTP-fluorescein, instead of UTP-DIG, use a 1/8000 dilution of the anti-fluorescein-AP antibody (BM 1426 338).
Post-antibody washes and histochemistry
1. Rinse 3 times with MABT. Transfer to scintillation vial.
2. Wash 3 x 1h with 10-20 ml MABT, by rolling. If desired, washing without rocking can proceed overnight for lower background.
3. Wash 3x10 min with NTMT.
4. Incubate with 1.5 ml NTMT + 4.5 µl/ml NBT + 3.5 µl/ml BCIP. Rock for first 20 min. (Develops faster at 37°C, if necessary)
5. When color has developed to the desired extent (30 min to 3 days), wash 3x with PTW. Refix in 4% HCHO/0.1% Glutaraldehyde/PTW, overnight, followed by PTW washes and storage in PTW/0.1% azide, at +4°C.
6. Clear in 50% glycerol/PTW then 80% glycerol/PTW/0.02% azide.
NTMT:
5M NaCl |
1 ml |
|
2M Tris-HCl pH 9.5 |
2.5 ml |
|
2M MgCl2 |
1.25 ml |
|
10% Tween-20 |
5 ml |
|
H20 |
40.25 ml |
|
Total |
50 ml |
Make from stocks on day of use.
N.B. Tween-20 final concentration is 1%.
References
Chitnis, A., Henrique, D., Lewis, J., Ish-Horowicz, D., and Kintner, C. (1995). Primary neurogenesis in Xenopus embryos regulated by a homologue of the Drosophila neurogenic gene Delta. Nature 375, 761-766.
Henrique, D., Adam, J., Myat, A., Chitnis, A., Lewis, J., and Ish-Horowicz, D. (1995). Expression of a Delta homologue in prospective neurons in the chick. Nature 375, 787-790.
Make 20% paraformaldehyde fresh before use: Heat 17 ml of water on a hot plate with stirring. Add 1 drop of 10 N NaOH, then 4 g of paraformaldehyde and stir until dissolved. Make up to 20 ml.
10X PBS (100 ml) 8 g NaCl, 0.2 g KCl, 1.15 g Na2HPO4, 0.2 g KH2PO4