Whole‐Mount In Situ Hybridization and Detection of RNAs in Vertebrate Embryos and Isolated Organs

Anne Pizard1, Anna Haramis2, Andrés E. Carrasco3, Paula Franco3, Silvia López3, Alejandra Paganelli3

1 Harvard Medical School, Boston, Massachusetts, 2 EMBL, Heidelberg, 3 University of Buenos Aires‐CONICET School of Medicine, Buenos Aires
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 14.9
DOI:  10.1002/0471142727.mb1409s66
Online Posting Date:  May, 2004
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Abstract

Nonisotopic in situ hybridization using intact embryos or organs is an important method for determining the spatial distribution of RNAs. Because it allows the analysis of large numbers of samples, it is amenable to temporal expression studies and comparison between different genotypes. It offers sensitivity and reproducibility. In addition, histological details are not lost during the staining process. The protocols in this unit can be used for whole‐mount in situ hybridization in Xenopus, mouse, and chicken embryos, as well as dissected organs from mouse and chicken. Preparation of digoxigenin‐labeled riboprobes is also described.

Keywords: in situ hybridization; gene expression; RNA; embryo

     
 
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Table of Contents

  • Basic Protocol 1: Whole‐Mount in situ Hybridization with Mouse or Chicken Embryos and Organs
  • Basic Protocol 2: Enzymatic Detection of RNA Hybrids in Mouse and Chicken Embryos and Organs
  • Alternate Protocol 1: Whole‐Mount in situ Hybridization with Mouse or Chicken Embryos and Organs
  • Alternate Protocol 2: Enzymatic Detection of RNA Hybrids in Mouse and Chicken Embryos and Organs
  • Support Protocol 1: Preabsorption of Fab Fragments with Embryonic Powder
  • Basic Protocol 3: Whole‐Mount in situ Hybridization with Xenopus Embryos
  • Basic Protocol 4: Detection of RNA Hybrids in Xenopus Embryos by Anti‐Digoxigenin Antibodies
  • Support Protocol 2: Synthesis of Digoxigenin‐Labeled RNA Probes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Whole‐Mount in situ Hybridization with Mouse or Chicken Embryos and Organs

  Materials
  • Mouse or chicken embryos or organs
  • PBS ( appendix 22), ice‐cold
  • 4% (w/v) paraformaldehyde in PBS (4% PFA; unit 14.1), 4°C
  • PBT (also see recipe): 0.1% or 1% (v/v) Tween 20 in PBS, 4°C and room temperature
  • 25%, 50%, and 75% methanol in PBT (methanol/PBT)
  • 100% methanol
  • 6% (v/v) hydrogen peroxide (H 2O 2; Aldrich) in PBT (optional)
  • 10 µg/ml proteinase K in PBT, not predigested
  • 2 mg/ml glycine (Merck) in PBT, freshly prepared
  • 0.2% (v/v) glutaraldehyde (EM grade, Sigma)/4% (w/v) paraformaldehyde in PBT, freshly prepared
  • Hybridization solution (see recipe)
  • Hybridization solution containing 1 µg/ml digoxigenin‐labeled riboprobe previously denaturated (see protocol 5)
  • Dissecting tools (e.g., scissors and forceps), wiped with 70% ethanol
  • Dissecting microscope
  • 6‐well culture plate with polycarbonate membrane inserts
  • 1.5‐ to 2‐ml microcentrifuge tubes
  • Heating block with adapter to hold 2‐ml microcentrifuge tubes
  • Rocker platform
NOTE: All washes are carried out in 10 ml (6‐well culture plates with polycarbonate membrane insert) or 1.5 to 2 ml (2‐ml microcentrifuge tubes) of solution at room temperature with gentle agitation, unless stated otherwise. Solutions should be warmed to the indicated temperature before use.

Basic Protocol 2: Enzymatic Detection of RNA Hybrids in Mouse and Chicken Embryos and Organs

  Materials
  • Hybridized samples (see protocol 1)
  • Solution I (see recipe), 70°C
  • Solution II (see recipe), 65°C
  • TBST (also see recipe): 0.1% (chick) or 1% (mouse) (v/v) Tween 20 in TBS (see recipe for TBS)
  • Blocking solution for chick: 10% heat‐inactivated sheep serum (see recipe) in TBST
  • Blocking solution for mouse: 10% heat‐inactivated sheep serum (see recipe), 0.1% blocking reagent (see recipe) in TBST
  • Alkaline phosphatase–conjugated Fab fragments of anti‐digoxigenin antibodies (Boehringer Mannheim), 4°C
  • Alkaline phosphatase buffer, pH 9.5 (NTMT; see recipe), freshly prepared
  • Reaction mix: 125 µg/ml BCIP and 250 µg/ml NBT in NTMT, pH 9.5
  • PBT buffer: 0.1% (chick) or 1% (mouse) Tween 20 in PBS
  • 4% (w/v) paraformaldehyde in PBS (4% PFA; unit 14.1) with 0.1% glutaraldehyde
  • PBS ( appendix 22)
  • 20%, 50%, and 80% glycerol/PBS solution
  • Rocker platform
  • Aluminum foil
  • 6‐well culture plate with polycarbonate membrane inserts

Alternate Protocol 1: Whole‐Mount in situ Hybridization with Mouse or Chicken Embryos and Organs

  Materials
  • Mouse or chicken embryos or organs
  • PBS ( appendix 22), ice cold
  • 4% (w/v) paraformaldehyde in PBS (4% PFA; unit 14.1), 4°C
  • PBT (also see recipe): 0.1% (v/v) Tween 20 in PBS, 4°C and room temperature
  • 25%, 50%, and 75% methanol in PBT (methanol/PBT)
  • 100% methanol
  • 6% (v/v) hydrogen peroxide (H 2O 2; Aldrich) in PBT (optional)
  • 10 µg/ml proteinase K in PBT, not predigested
  • 2 mg/ml glycine (Merck) in PBT, freshly prepared
  • 0.2% (v/v) glutaraldehyde (EM grade, Sigma)/4% (w/v) paraformaldehyde in PBT, freshly prepared
  • Prehybridization solution A (see recipe), 65°C
  • Hybridization solution A: prehybridization solution A (see recipe) containing 1 µg/ml digoxigenin‐labeled riboprobe (see protocol 5)
  • Dissecting tools (e.g., scissors and forceps), wiped with 70% ethanol
  • Dissecting microscope
  • 20‐ml snap‐cap glass vials
  • Heating block with adapter to hold 2‐ml microcentrifuge tubes
  • Platform rocker
NOTE: All washes are carried out in 5 to 10 ml (20‐ml glass vials) or 1.5 to 2 ml (2‐ml microcentrifuge tubes) of solution for 5 min at room temperature with gentle agitation, unless stated otherwise. Solutions should be warmed to the indicated temperature before use.

Alternate Protocol 2: Enzymatic Detection of RNA Hybrids in Mouse and Chicken Embryos and Organs

  Materials
  • Hybridized mouse or chicken embryo or organ sample in hybridization solution A (see protocol 3)
  • Prehybridization solution A (see recipe), 70°C
  • 2× SSC, pH 4.5 (see recipe for 20× SSC), 70°C
  • 0.1% (v/v) CHAPS (3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate)/2× SSC, 70°C
  • 20 µg/ml RNase A (Boehringer Mannheim) in 0.1% CHAPS/2× SSC, 37°C
  • Maleic acid buffer (MAB; see recipe), 70°C and room temperature
  • PBS ( appendix 22)
  • PBT (also see recipe): 0.1% (v/v) Tween 20 in PBS
  • Blocking solution (see recipe)
  • Alkaline phosphatase–conjugated Fab fragments of anti‐digoxigenin antibodies (Boehringer Mannheim), preabsorbed (see protocol 8), 4°C
  • 0.1% (w/v) BSA in PBT
  • Alkaline phosphatase buffer, pH 9.5 (NTMT; see recipe), freshly prepared
  • 2 mM levamisole (Sigma) in NTMT (optional)
  • NBT/BCIP substrate solution (unit 14.7) or BM purple AP substrate (Boehringer Mannheim)
  • Plastic Pasteur pipets
  • 70°C heating block with adapter for 2‐ml microcentrifuge tubes
  • Rocker platform
  • 37° and 70°C water bath (optional)
  • 20‐ml snap‐cap glass vials
  • Aluminum foil
  • Dissecting microscope equipped with a camera
CAUTION: Formamide and DEPC are hazardous; see manufacturer's guidelines for handling, storage, and disposal.NOTE: All incubations and washes are carried out at the temperatures indicated and with gentle rocking (see protocol 1, step ). Solutions should be warmed to the indicated temperature before use.

Support Protocol 1: Preabsorption of Fab Fragments with Embryonic Powder

  Materials
  • Embryos or organs under study (e.g., embryonic day‐10 to ‐12 mouse embryos or stage‐24 to ‐26 chicken embryos)
  • Liquid nitrogen
  • Acetone, ice cold
  • PBT (also see recipe): 0.1% Tween 20 in PBS ( appendix 22 for PBS)
  • BSA
  • Sheep serum (see recipe), heat‐inactivated
  • Alkaline phosphatase–conjugated Fab fragment of anti‐digoxigenin antibody (Boehringer Mannheim)
  • Blocking solution (see recipe)
  • Mortar and pestle
  • 50‐ml centrifuge tubes
  • Beckman TJ‐6 centrifuge or equivalent, 4°C
  • Filter paper
  • Air‐tight microcentrifuge tubes
  • Rocker platform

Basic Protocol 3: Whole‐Mount in situ Hybridization with Xenopus Embryos

  Materials
  • Albino Xenopus laevis embryos
  • 2% (w/v) cysteine, pH 7.8
  • MEMFA buffer (see recipe)
  • PBS ( appendix 22)
  • 50% and 75% ethanol in sterile distilled water
  • 100% ethanol
  • 25% methanol/PBT
  • PBT (also see recipe): 0.1% (v/v) Tween 20 in PBS, pH 7.8
  • 2.5 µg/ml proteinase K in PBT
  • 0.1 M triethanolamine (TEA) buffer in PBT, pH 7.8 (unit 14.3, except use PBT), prepared fresh
  • Acetic anhydride (Sigma)
  • 4% formaldehyde in PBT
  • Prehybridization solution B (see recipe)
  • Hybridization solution B: prehybridization solution B (see recipe) containing 1 µg/ml digoxigenin‐labeled riboprobe (see protocol 8)
  • 2‐ml screw‐cap plastic conical tubes
  • 37° and 60°C shaking water bath
CAUTION: All solutions should be treated with diethylpyrocarbonate (DEPC; see unit 4.1) and autoclaved to inhibit RNase activity. DEPC is a suspected carcinogen and should be handled carefully.NOTE: The samples should always be kept covered with liquid to avoid drying, which causes high levels of background staining. Therefore, when exchanging solutions, always leave the sample covered by a small quantity of liquid. This also prevents physical damage or loss of samples.NOTE: It is important that the embryos be kept in suspension during the early fixation steps, otherwise they will flatten.

Basic Protocol 4: Detection of RNA Hybrids in Xenopus Embryos by Anti‐Digoxigenin Antibodies

  Materials
  • Hybridized Xenopus laevis embryos (see protocol 6)
  • Prehybridization solution B (see recipe), 60°C
  • 2× and 0.2× SSC (see recipe for 20×), 60°C and room temperature
  • 2× SSC containing 20 µg/ml RNase A and 10 U/ml RNase T1 (see recipes)
  • 0.1 (v/v) DEPC
  • Maleic acid buffer (MAB; see recipe)
  • 2% (w/v) blocking reagent (see recipe) in MAB
  • Alkaline phosphatase–conjugated Fab fragments of anti‐digoxigenin antibody (Boehringer Mannheim) diluted 1:2000 in 2% blocking reagent (see protocol 5)
  • Alkaline phosphatase buffer, pH 9.5 (NTMT; see recipe) containing 5 mM levamisole (Sigma), filtered
  • NBT/BCIP substrate solution (unit 14.7) or BM purple AP substrate (Boehringer Mannheim)
  • Ethanol
  • 4% (w/v) formaldehyde in PBT
  • 37° and 60°C shaking water baths
  • Aluminum foil
  • Dissecting microscope equipped with a camera
CAUTION: Dimethylformamide and DEPC are hazardous; see manufacturer's guidelines for handling, storage, and disposal.NOTE: All solutions should be treated with diethylpyrocarbonate (DEPC; see unit 4.1) and autoclaved to inhibit RNase activity.NOTE: The samples should always be kept covered with liquid to avoid drying, which causes high levels of background staining. Therefore, when exchanging solutions, always leave the sample covered by a small quantity of liquid. This also prevents physical damage or loss of samples.

Support Protocol 2: Synthesis of Digoxigenin‐Labeled RNA Probes

  Materials
  • Distilled water, sterile
  • 10× transcription buffer: 400 mM Tris·Cl (pH 8.25)/60 mM MgCl 2/20 mM spermidine (Boehringer Mannheim)
  • Nucleotide mix (pH 8): 10 mM GTP/10 mM ATP/10 mM CTP/6.5 mM UTP/ 3.5 mM digoxigenin‐UTP (Boehringer Mannheim)
  • 1 µg/ml linearized plasmid (unit 4.7)
  • 40 U/ml placental ribonuclease inhibitor (RNasin, Boehringer Mannheim)
  • 20 U/µl SP6, T3, or T7 RNA polymerase
  • RNase‐free DNase I (unit 4.1)
  • TE buffer, pH 8 ( appendix 22) containing 0.1% SDS, DEPC‐treated
  • Sephadex G‐25 (coarse)
  • 100% ethanol
  • 3 M sodium acetate, pH 5.2 ( appendix 22)
  • 70% ethanol in DEPC‐treated water (see unit 4.1)
  • RNA loading buffer (see recipe)
  • 37°C water bath
  • 1‐ml syringe
  • Additional reagents and equipment for agarose gel electrophoresis (unit 2.5)
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Figures

Videos

Literature Cited

Literature Cited
   Carrasco, A.E. and Malacinski, G. 1987. Localization of Xenopus homeo‐box gene transcripts during embryogenesis and in the adult nervous system. Dev. Biol. 121:69‐81.
   Crossley, P.H. and Martin, G.R. 1995. The mouse Fgf8 gene encodes a family of polypeptides and is expressed in regions that direct outgrowth and patterning in the developing embryo. Development 121:439‐451.
   Franco, P., Paganelli, A., López, S., and Carrasco, A.E. 1999. Functional association of retinoic acid and hedgehog signaling in Xenopus primary neurogenesis. Development 126:4257‐4265.
   Haramis, A.G., Brown, J.M., and Zeller, R. 1995. The limb deformity mutation disrupts the SHH/FGF‐4 feedback loop and regulation of 5′ HoxD genes during limb pattern formation. Development 121:4237‐4245.
   Harland, R.M. 1991. In situ hybridization: An improved whole‐mount method for Xenopus embryos. Methods Cell Biol. 36:685‐695.
   Hauptmann, G. and Gerster, T. 1994. Two‐color whole‐mount in situ hybridization to vertebrate and Drosophila embryos. Trends Genet. 10:266.
   Jowett, T. and Lettice, L. 1994. Whole mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin‐ and fluorescein‐ labeled probes. Trends Genet. 10:73‐74.
   Lamb, T.M., Knecht, A.K., Smith, W.C., Stachel, S.E., Economides, A.N., Stahl, N., Yancopolous, G.D., and Harland, R.M. 1993. Neural induction by the secreted polypeptide noggin. Science 262:713‐718.
   López, S., Paganelli, A., Rosato Siri, M., Ocaña, O., Franco, P., and Carrasco, A.E. 2003. Notch activates sonic hedgehog and both are involved in the specification of dorsal midline cell‐fates in Xenopus. Development 130:2225‐2238.
   Paganelli, A., Ocaña, O., Prat, M., Franco, P., López, S., Morelli, L., Adamo, A., Riccomagno, M., Matssubara, E., Shoji, M., Affranchino, J., Castaño, E., and Carrasco, A.E. 2001. The Alzheimer‐related gene presenilin‐1 facilitates sonic hedgehog signaling in Xenopus primary neurogenesis. Mechan. Dev. 107:119‐131.
   Riddle, R.D., Johnson, R.L., Laufer, E., and Tabin, C. 1993. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75:1401‐1416.
   Rosen, B. and Beddington, S.P. 1993. Whole mount in situ hybridization in the mouse embryo: Gene expression in three dimensions. Trends Genet. 9:162‐167.
   Schulte‐Merker, S. 1993. In situ hybridization. In The Zebrafish Book, 2nd ed. (M. Westerfield, ed.) pp. 9.16‐9.21. University of Oregon Press, Eugene, Oreg.
   Sive, H.L., Grainger, R.M., and Harland, R.M. (eds.) 1994. Early Development of Xenopus laevis, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
   Tautz, D. and Pfeifle, C. 1989. A nonradioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98:81‐85.
   Wilkinson, D.G. 1993. In situ hybridization. In Essential Development Biology. A Practical Approach (C.D. Stern, and, P.W.H. Holland, eds.) pp. 257‐276. IRL Press, Oxford.
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