Hybridization Histochemistry of Neural Transcripts

W. Scott Young1, Éva Mezey1

1 National Institutes of Health, Bethesda, Maryland
Publication Name:  Current Protocols in Neuroscience
Unit Number:  Unit 1.3
DOI:  10.1002/0471142301.ns0103s25
Online Posting Date:  February, 2004
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Expression of genes is manifested by the production of RNA transcripts within cells. Hybridization histochemistry (or in situ hybridization) permits localization of these transcripts with cellular resolution or better. Furthermore, the relative amounts of transcripts detected within different tissues or the same tissues in different states (e.g., physiological or developmental) may be quantified. This unit describes hybridization histochemical techniques using either oligonucleotide probes or RNA probes (riboprobes). Also presented is the use of probes labeled with digoxigenin for colorimetric detection of RNA transcripts and a technique to detect the Y chromosome using either mouse or human riboprobes. Finally, a procedure is presented for the autoradiographic detection of radiolabeled probes. Methods are provided for labeling oligodeoxynucleotide and RNA probes and performing northern analyses using these probes.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Hybridization Histochemistry with Oligodeoxynucleotide Probes
  • Basic Protocol 2: Hybridization Histochemistry with RNA Probes
  • Basic Protocol 3: Detection of Digoxigenin‐Labeled Probes Using AP‐Conjugated Anti‐Digoxigenin Antibodies
  • Alternate Protocol 1: Tyramide Signal Amplification (TSA) Detection of Digoxigenin‐Labeled Probes
  • Basic Protocol 4: Detection of Chromosomal DNA (Y Chromosome) Using Riboprobes
  • Basic Protocol 5: Detection of Radiolabeled Probes
  • Support Protocol 1: Preparation of Oligonucleotide Probes for Hybridization Histochemistry
  • Support Protocol 2: Preparation of RNA Probes (Riboprobes) for Hybridization Histochemistry
  • Support Protocol 3: Northern Analysis Using Oligodeoxynucleotide Probes and Riboprobes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Hybridization Histochemistry with Oligodeoxynucleotide Probes

  Materials
  • 50% (v/v) formamide in 4× SSPE ( appendix 2A)
  • Slides containing 12‐µm tissue sections prepared by cryostat, mounted on Superfrost Plus slides (Fisher), and defatted (unit 1.1)
  • Hybridization solution (see recipe) containing ∼1 × 106 dpm/50 µl 35S‐labeled oligodeoxynucleotide probe or 1 to 5 µl/50 µl digoxigenin‐labeled oligonucleotide probe (see Mezey et al., protocol 7)
  • 1× SSPE/1 mM DTT, room temperature and 55°C
  • 70% ethanol
  • Sterile Bio‐Assay dishes (Nunc; 245 × 245 × 30 mm)
  • Whatman 3MM chromatography paper
  • Glass coverslips
  • Staining dishes and tubs
  • Slide rack
  • 55°C water bath
NOTE: Use DEPC‐treated water ( appendix 2A) for all reagents in pretreatment and hybridization steps.

Basic Protocol 2: Hybridization Histochemistry with RNA Probes

  Materials
  • 50% (v/v) formamide in 4× SSPE ( appendix 2A)
  • Slides containing 12‐µm tissue sections prepared by cryostat, mounted on Superfrost Plus slides (Fisher), and defatted (unit 1.1)
  • Hybridization solution (see recipe) containing ∼1 × 106 dpm/50 µl 35S‐labeled riboprobe or 3 to 10 µl/100 µl digoxigenin‐labeled riboprobe (see protocol 8)
  • 4× SSPE/1 mM DTT
  • RNase A solution (see recipe), 37°C
  • 0.1× SSPE/1 mM DTT, room temperature and 65°C
  • 1× SSPE
  • 70% ethanol
  • Sterile Bio‐Assay dishes (Nunc; 245 × 245 × 30 mm)
  • Whatman 3MM chromatography paper
  • Glass coverslips
  • Staining dishes and tubs
  • Slide rack
  • 55°C incubator
  • 65°C water bath
NOTE: Use DEPC‐treated water ( appendix 2A) for all reagents in pretreatment and hybridization steps.

Basic Protocol 3: Detection of Digoxigenin‐Labeled Probes Using AP‐Conjugated Anti‐Digoxigenin Antibodies

  Materials
  • Digoxigenin‐labeled sections on slides (see protocol 1, step , or protocol 2, step )
  • TBS, pH 7.5 ( appendix 2A; room temperature)
  • Detection buffer (see recipe)
  • Alkaline phosphatase–conjugated sheep polyclonal anti‐digoxigenin antibody (Roche)
  • Development buffer (see recipe)
  • NBT/BCIP substrate working solution (see recipe)
  • 1× SSPE ( appendix 2A)
  • Cytoseal 60 (Stephens Scientific) or similar organic‐basic mounting medium
  • Staining dishes or tubs
  • Slide warmer

Alternate Protocol 1: Tyramide Signal Amplification (TSA) Detection of Digoxigenin‐Labeled Probes

  • Peroxidase‐conjugated sheep polyclonal anti‐digoxigenin antibody (Roche)
  • Renaissance TSA‐Indirect kit (Perkin‐Elmer) containing:
    • 2× diluent
    • Blocking reagent
    • Biotinylated tyramide
  • Streptavidin–Texas red or streptavidin‐fluorescein conjugate (Perkin‐Elmer)
  • Streptavidin‐HRPO conjugate (Perkin‐Elmer)
  • TBS, pH 8.0 ( appendix 2A; room temperature)
  • Diaminobenzidine (DAB) tablets (Sigma)
  • Urea/hydrogen peroxide tablets (Sigma)
  • Streptavidin–alkaline phosphatase conjugate (Jackson Immunoresearch)
  • Cytoseal 60 (Stephens Scientific) or similar organic‐basic mounting medium

Basic Protocol 4: Detection of Chromosomal DNA (Y Chromosome) Using Riboprobes

  Materials
  • Slides containing defatted 12‐µm fresh‐frozen or paraffin‐embedded sections prepared by cryostat (unit 1.1) or whole fixed cells
  • Paraformaldehyde–picric acid solution (see recipe)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 0.1×, 0.5×, 1×, and 2× SSC ( appendix 2A)
  • Citrisolv (Fisher) or xylene
  • 70%, 80%, 95%, and 100% ethanol
  • Citra Plus, pH 6 (BioGenex)
  • Optional: Reagents for combined immunocytochemistry/immunohistochemistry including:
    • Blocking solution: PBS containing 1% (w/v) BSA and 0.6% (v/v) Triton X‐100
    • Biotinylated secondary antibody (from Vectastain ABC kit, Vector Laboratories, or Jackson Immunoresearch)
    • 0.1 M Tris⋅Cl, pH 8 ( appendix 2A)
    • Streptavidin‐HRPO conjugate (Perkin‐Elmer)
    • Biotinylated tyramide (Perkin‐Elmer)
    • Fluorochrome‐labeled streptavidin of different color from that used to develop the hybridization histochemical signal
  • Hybridization solution (see recipe) containing 3 to 10 µl/100 µl digoxigenin‐labeled riboprobe ( protocol 8) to a mouse repeat sequence (Bishop and Hatat, ; Mezey et al., ) or human Y‐chromosomal marker DYZ1 (Mezey et al., )
  • 70% formamide/2× SSC
  • Plastic Coplin jar
  • Staining dishes and tubs
  • Slide rack
  • Sterile Bio‐Assay dishes (Nunc, 245 × 245 × 30 mm)
  • 55°, 65°, and 80°C incubators
  • 65° and 80°C water bath

Basic Protocol 5: Detection of Radiolabeled Probes

  Materials
  • Ilford K5.D or Kodak NTB‐3 nuclear emulsion
  • 7.5 M ammonium acetate
  • 35S‐labeled samples, slide‐mounted (see protocol 1 and protocol 2)
  • Kodak D‐19 photographic developer, 17°C
  • Kodak Rapid Fix (without hardener), 17°C
  • Counterstain (optional): 0.4% toluidine blue, 2 µg/ml ethidium bromide, hematoxylin/eosin, or other stain of choice
  • Cytoseal 60 (Stephens Scientific) or similar organic‐based mounting medium
  • Darkroom with safelight
  • Coplin jars
  • Spatula
  • 40°C water bath
  • Black slide boxes
  • Desiccant capsules (e.g., Humi‐caps from United Desiccants–Gates)
  • Black photography tape
  • Slide racks
  • Slide warmer

Support Protocol 1: Preparation of Oligonucleotide Probes for Hybridization Histochemistry

  Materials
  • 5× tailing buffer (see recipe)
  • Oligonucleotide to be used as probe
  • [α‐35S]dATP (>1000 Ci/mmol; Perkin‐Elmer)
  • Terminal deoxynucleotidyl transferase (TdT; Roche or Invitrogen)
  • 250 µM digoxigenin‐dUTP/1 mM dNTP (or dATP) mix (ingredients available from Roche; store mix indefinitely at −20°C)
  • TE buffer, pH 7.6 ( appendix 2A)
  • 4 M NaCl
  • 25 µg/µl yeast tRNA
  • 70% and 100% ethanol
  • 5 M dithiothreitol (DTT)

Support Protocol 2: Preparation of RNA Probes (Riboprobes) for Hybridization Histochemistry

  Materials
  • Linearized plasmid containing cDNA to be transcribed
  • [α‐35S]UTP (>1000 Ci/mmol; Perkin‐Elmer)
  • 5× transcription buffer (see recipe)
  • 100 mM dithiothreitol (DTT)
  • 10 mM ATP, CTP, and GTP
  • RNasin (Promega)
  • 10 to 20 U/µl RNA polymerase (SP6, T3, or T7)
  • 1 mM UTP/4 mM digoxigenin‐11‐UTP mix (ingredients available from Roche; store mix indefinitely at −20°C)
  • 1 U/µl RNase‐free DNase I ( appendix 2A)
  • TE buffer ( appendix 2A)
  • 4 M NaCl
  • 25 µg/µl yeast tRNA
  • 70% and 100% ethanol
  • 5 M DTT
  • 10% (w/v) sodium dodecyl sulfate (SDS)
  • TE buffer/5% (w/v) SDS
NOTE: Use DEPC‐treated water ( appendix 2A) for all reagents.

Support Protocol 3: Northern Analysis Using Oligodeoxynucleotide Probes and Riboprobes

  Materials
  • RNA sample
  • GeneScreen membrane (Perkin‐Elmer)
  • Northern hybridization solution (see recipe)
  • Northern hybridization solution containing ∼2 × 106 dpm/ml 35S‐labeled oligodeoxynucleotide probe (see protocol 7) or riboprobe (see protocol 8)
  • 1× SSPE ( appendix 2A)/0.2% SDS, room temperature and 55°C (for oligodeoxynucleotide probes)
  • 0.1× SSPE/0.2% SDS, 65°C (for riboprobes)
  • UV cross‐linker (e.g., Stratalinker from Stratagene)
  • 70°C vacuum oven
  • Plastic wrap
  • Autoradiography cassettes
  • Intensifying screens
  • Autoradiography film (e.g., Kodak Biomax MR)
NOTE: Use DEPC‐treated water ( appendix 2A) for all reagents.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

  •   FigureFigure 1.3.1 Photomicrograph shows oxytocin and vasopressin neurons within the human supraoptic nucleus labeled respectively, with 35S‐labeled and digoxigenin‐labeled probes. The arrow indicates a neuron containing both transcripts. Neurons containing the digoxigenin‐labeled probe show a dark stain from development of the alkaline phosphatase on the anti‐digoxigenin antibodies. The deposition of the radiolabeled probe is indicated by the gray‐colored silver grains. Note that a nucleus (n) is clearly delineated by the alkaline phosphatase staining.

Videos

Literature Cited

Literature Cited
   Albertson, D.G., Fishpool, R.M., and Birchall, P.S. 1995. Fluorescence in situ hybridization for the detection of DNA and RNA. Methods Cell Biol. 48:339‐364.
   Bishop, C.E. and Hatat, D. 1987. Molecular cloning and sequence analysis of a mouse Y chromosome RNA transcript expressed in the testis. Nucl. Acids Res. 15:2959‐2969.
   Bobrow, M.N., Harris, T.D., Shaughnessy, K.J., and Litt, G.J. 1989. Catalyzed reporter deposition: A novel method of signal amplification. J. Immunol. Methods 125:279‐285.
   Bradley, D.J., Towle, H.C., and Young, W.S. III. 1992. Spatial and temporal expression of α and β thyroid hormone receptor mRNAs, including the β2 subtype, in the developing mammalian nervous system. J. Neurosci. 12:2288‐2302.
   Burgunder, J.‐M. and Young, W.S. III. 1988. The distribution of thalamic projection neurons containing cholecystokinin messenger RNA, using in situ hybridization histochemistry and retrograde labeling. Mol. Brain Res. 4:179‐189.
   Gerfen, C.R. 1989. Quantification of in situ hybridization histochemistry for analysis of brain function. In Methods in Neuroscience (P.M. Conn, ed.) pp. 79‐97. Academic Press, New York.
   Hunyady, B., Krempels, K., Harta, G., and Mezey, E. 1996. Immunohistochemical signal amplification by catalyzed reporter deposition and its application in double immunostainings. J. Histochem. Cytochem. 12:1353‐1362.
   Kerstens, H.M.J., Poddihe, P.J., and Hanselaar, A.G.J.M. 1995. A novel in situ hybridization signal amplification method based on the deposition of biotinylated tyramine. J. Histochem. Cytochem. 43:347‐352.
   Kiyama, H. and Emson, P.C. 1991. An in situ hybridization histochemistry method for the use of alkaline phosphatase–labeled oligonucleotide probes in small intestine. J. Histochem. Cytochem. 39:1377‐1384.
   Mezey, É., Chandross, K.J., Harta, G., Maki, R.A., and McKercher, S.R. 2000. Turning blood into brain: Cells bearing neuronal antigens generated in vivo from bone marrow. Science 290:1775‐1779.
   Mezey, É., Key, S., Vogelsang, G., Szalayova, I., and Lange, G.D., and Crain, B. 2003a. Transplanted bone marrow generates new neurons in human brains, Proc. Nat. Acad. Sci. U.S.A. 100:1364‐1369.
   Mezey, É., Nagy, A., Szalayova, I., Key, S., Bratincsak, A., Baffi, J., and Shahar, T. 2003b. Comment on “Failure of bone marrow cells to transdifferentiate into neural cells in vivo.” Science 299:1184.
   Morey, A.L. 1995. Non‐isotopic in situ hybridization at the ultrastructural level. J. Pathol. 176:113‐121.
   Rogers, A.W. 1979. Techniques of Autoradiography. Elsevier Science Publishing, New York.
   Romijn, H.J. and van Uum, J.F.M. 1999. Double immunolabeling of neuropeptides in the human hypothalamus as analyzed by confocal laser scanning fluorescence microscopy. J. Histochem. Cytochem. 47:229‐235.
   Rosen, B. and Beddington, R.S. 1993. Whole‐mount in situ hybridization in the mouse embryo: Gene expression in three dimensions. Trends Genet. 9:162‐167.
   Swiger, R.R. and Tucker, J.D. 1996. Fluorescence in situ hybridization: A brief review. Environ. Mol. Mutagen. 27:245‐254.
   Theise, N.D., Krause, D.S., and Sharkis, S. 2003. Comment on “Little evidence for developmental plasticity of adult hematopoietic stem cells.” Science 299:1317
   Tran, S.D., Pillemer, S.R., Dutra, A., Barrett, A.J., Brownstein, M.J., Key, S., Pak, E., Leakan, R.A., Yamada, K.M., Baum, B.J., and Mezey, É., 2003. Human bone marrow‐derived cells differentiate into buccal epithelial cells in vivo without fusion. Lancet 361: 1084‐1088.
   Valentino, K.L., Eberwine, J.H., and Barchas, J.D. (eds.) 1987. In Situ Hybridization: Applications to Neurobiology. Oxford University Press, New York.
   Westlake, T.M., Howlett, A.C., Bonner, T.I., Matsuda, L.A., and Herkenham, M. 1994. Cannabinoid receptor binding and messenger RNA expression in human brain: An in vitro receptor autoradiography and in situ hybridization histochemistry study of normal aged and Alzheimer's brains. Neuroscience 63:637‐652.
   Wilkinson, D.G. (ed.) 1992. In Situ Hybridization. A Practical Approach. Oxford University Press, New York.
   Young, W.S. III 1992. In situ hybridization with oligodeoxyribonucleotide probes. In In Situ Hybridization: A Practical Approach (D.G. Wilkinson, ed.) pp. 33‐44. Oxford University Press, New York.
   Young, W.S. III, Mezey, É., and Siegel, R.E. 1986. Vasopressin and oxytocin mRNAs in adrenalectomized and Brattleboro rats: Analysis by quantitative in situ hybridization histochemistry. Mol. Brain Res. 1:231‐241.
   Young, W.S. III, Reynolds, K., Shepard, E.A., Gainer, H., and Castel, M. 1990. Cell‐specific expression of the rat oxytocin gene in transgenic mice. J. Neuroendocrinol. 2:917‐925.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library