In Situ Hybridization Histochemistry

Zaal Kokaia1

1 University Hospital, Lund
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 2.7
DOI:  10.1002/0471140856.tx0207s05
Online Posting Date:  May, 2001
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Abstract

This unit describes two methods of in situ hybridization: one uses an 35S‐labeled oligonucleotide probe and the other uses a digoxigenin‐labeled oligonucleotide probe on frozen, cryostat‐sectioned samples. These methods allow detection of the physical distribution and expression levels of target mRNA. Protocols are also included for labeling the probes and preparing the sample material.

     
 
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Table of Contents

  • Basic Protocol 1: In Situ Hybridization with Radioactive Probes
  • Alternate Protocol 1: In Situ Hybridization with Nonradioactive Probes
  • Support Protocol 1: 3′ Labeling Oligonucleotide Probes with [35S]dATP
  • Support Protocol 2: 3′ Labeling of Oligonucleotide Probes with Digoxigenin
  • Support Protocol 3: Preparation of Tissue
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Situ Hybridization with Radioactive Probes

  Materials
  • Hybridization buffer (see recipe)
  • 10 mg/ml salmon testes DNA
  • 1000 Ci/mmol [35S]dATP‐radiolabeled probe (see protocol 3)
  • 5 M DTT (see recipe)
  • Glass slide with fixed and dehydrated specimen of interest (see protocol 5)
  • 1× SSC (see recipe), 55°C
  • 70%, 80%, 95%, and 100% ethanol
  • D19 X‐ray film developer (Kodak)
  • X‐ray film fixer (Kodak or equivalent)
  • K5 photoemulsion (Ilford)
  • AGFA G333 fix
  • Cresyl violet stock solution: 5 g cresyl violet in 1 liter H 2O
  • 10% (v/v) acetic acid
  • Xylene
  • Mounting medium (DPX, Permount, or Petrex)
  • 1.5‐ml microcentrifuge tubes, sterile
  • Humidified chamber: box with tight lid and wet filter paper placed to prevent contact with slides
  • 42°C incubator
  • 40°C and 55°C water bath
  • Staining dish and slide racks
  • Autoradiographic 14C microscale (Amersham)
  • Hyperfilm β‐max X‐ray film (Amersham)
  • Slide box
  • Black electrician's tape
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area following the guidelines provided by your local radiation safety offices (also see appendix 1A).

Alternate Protocol 1: In Situ Hybridization with Nonradioactive Probes

  Materials
  • DIG‐labeled probe ( protocol 4)
  • Hybridization buffer (see recipe)
  • Glass slides with fixed and dehydrated specimen
  • 1× SSC (see recipe), 48°C
  • Binding buffer (see recipe), prepare fresh
  • Anti‐DIG‐alkaline phosphatase–labeled antibody (Roche Molecular Biochemicals)
  • Normal sheep serum
  • Triton X‐100
  • Wash buffer (see recipe), prepare fresh
  • Color solution (see recipe)
  • Stop buffer (see recipe), prepare fresh
  • Mounting medium, aqueous (Aquamount, BDH)
  • Humidified chamber: box with tight lid and wet filter paper placed to prevent contact with slides
  • Slide racks and staining dishes
  • 48° and 55°C water baths

Support Protocol 1: 3′ Labeling Oligonucleotide Probes with [35S]dATP

  Materials
  • 10 U/µl terminal deoxyribonucleotidyl transferase (TdT; Amersham Pharmacia Biotech)
  • TdT reaction buffer (supplied with TdT enzyme; also see recipe)
  • DEPC‐treated H 2O (see recipe)
  • 10 mCi/ml [35S]dATP (1000 Ci/mmol)
  • Oligonucleotide to be labeled, ∼50‐mer
  • Absolute methanol (HPLC grade)
  • 0.1 M Tris⋅Cl, pH 8.0 (see recipe)
  • 20% ethanol, ice‐cold
  • Scintillation fluid
  • 5 M DTT (see recipe)
  • 1.5‐ml microcentrifuge tubes, sterile
  • NENSORB column (DuPont NEN Research Products)
  • 10‐ml syringe
  • Scintillation vial
CAUTION: When working with radioactivity, take appropriate precautions to avoid contamination of the experimenter and surroundings. Carry out the experiment and dispose of wastes in an appropriately designated area following the guidelines provided by your local radiation safety offices (also see appendix 1A).

Support Protocol 2: 3′ Labeling of Oligonucleotide Probes with Digoxigenin

  Materials
  • 5× DIG labeling buffer (see recipe)
  • DEPC‐treated H 2O (see recipe)
  • 25 mM CoCl 2
  • 1 mM DIG‐dUTP (Roche Molecular Biochemicals)
  • Oligonucleotide
  • 10 U/µl terminal deoxyribonucleotidyl transferase (TdT)
  • 1.5‐ml microcentrifuge tubes, sterile

Support Protocol 3: Preparation of Tissue

  Materials
  • 4% paraformaldehyde (see recipe)
  • Phosphate buffered saline (PBS; see recipe)
  • 70%, 80%, and 90% ethanol
  • Poly‐L‐lysine or chrome alum coated slides (see recipe)
  • Additional materials and reagents for cryosectioning (Watkins, ).
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Figures

Videos

Literature Cited

Literature Cited
   Cassela, V.P., Kay, J., and Lawson, S.J. 1997. The Rat Nervous System. An Introduction to Preparatory Techniques. John Wiley & Sons, West Sussex.
   Emson, P.C. 1993. In‐situ hybridization as methodological tool for the neuroscientist. Trends Neurosci. 16:9‐16.
   John, H., Birnstiel, M., and Jones, K. 1969. RNA‐DNA hybrids at the cytological level. Nature 223:582‐587.
   Pardue, M.L. and Gall, J.G. 1969. Molecular hybridization of radioactive DNA to the DNA of cytological preparations. Proc. Natl. Acad. Sci. U.S.A. 64:600‐604.
   Watkins, S. 1989. Cryosectioning. In Current Protocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl, eds.) pp. 14.2.1‐14.2.8. John Wiley & Sons, New York
   Wilcox, J.N. 1993. Fundamental principles of in situ hybridization. J. Histochem. Cytochem. 41:1725‐1733.
   Wilkinson, D.G. 1992. The theory and practice of in situ hybridization. In In Situ Hybridization. A Practical Approach (D.G. Wilkinson ed.) pp. 1‐13. Oxford University Press, New York.
   Young, W.S., III. 1990. In situ hybridization histochemistry. In Handbook of Chemical Neuroanatomy, Vol. 8: Analysis of Neuronal Microcircuits and Synaptic Interactions (A. Björklund, T. Hökfelt, F.G. Wouterlood, and A.N. van den Pol, eds.) pp. 481‐512. Elsevier Science Publishers, New York.
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