, Volume 100, Issue 6, pp 431–440 | Cite as

A single protocol to detect transcripts of various types and expression levels in neural tissue and cultured cells: in situ hybridization using digoxigenin-labelled cRNA probes

  • Nicole Schaeren-Wiemers
  • Andrea Gerfin-Moser


We have developed a simple non-radioactive in situ hybridization procedure for tissue sections and cultured cells using digoxigenin-labelled cRNA probes. This protocol can be applied for the detection of various transcripts present at a wide range of expression levels in the central nervous system. Cerebellar hybridization signals for transcripts estimated to be expressed at high (MBP, myelin basic protein), moderate (GluR1, subunit of AMPA/kainate sensitive glutamate receptors) and low (inositol polyphosphate-5-phosphatase) levels of abundance are demonstrated as examples. The sensitivity and cellular resolution were significantly improved by avoiding any ethanol treatment commonly used in other procedures. The localization of a labelled cell with respect to its environment is shown to be more easily assessed by counterstaining of the tissue with the nuclear dye Hoechst 33258. The present protocol can be combined with immunocytochemistry as demonstrated for glial fibrillary acidic protein (GFAP). All steps of the procedure, including preparation and labelling of the cRNA probes, pretreatment of tissue, hybridization and visualization of the labelled transcripts, are described in detail.


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Angerer LM, Angerer RC (1991) Localization of mRNAs by in situ hybridization. Methods Cell Biol 35:37–71Google Scholar
  2. Bachmann S, Le Hir M, Eckardt K-U (1993) Co-localization of erythropoietin mRNA and ecto-5′-nucleotidase immunoreactivity in peritubular cells of rat renal cortex indicates that fibroblasts produce erythropoietin. J Histochem Cytochem 41:335–341Google Scholar
  3. Berridge MJ, Irvine RF (1989) Inositol phosphates and cell signalling. Nature 341:197–205Google Scholar
  4. Biffo S, Verdun di Cantogno L, Fasolo A (1992) Double labeling with non-isotopic in situ hybridization and BrdU immunohistochemistry: calmodulin (CaM) mRNA expression in postmitotic neurons of the olfactory system. J Histochem Cytochem 40:535–540Google Scholar
  5. Boehringer Mannheim (1992) Nonradioactive in situ hybridization: application manual. Boehringer Mannheim GmbH Biochemica, Mannheim, GermanyGoogle Scholar
  6. Colman DR, Kreibich G, Frey AB, Sabatini DD (1982) Synthesis and incorporation of myelin peptides into CNS myelin. J Cell Biol 95:598–608Google Scholar
  7. Dörries U, Bartsch U, Nolte Ch, Roth J, Schachner M (1993) Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies. Histochemistry 99:251–262Google Scholar
  8. Emson PC (1993) In-situ hybridization as a methodological tool for the neuroscientist. Trends Neurol Sci 16:9–16Google Scholar
  9. Hollmann M, O'Shea-Greenfield A, Rogers SW, Heinemann S (1989) Cloning by functional expression of a member of the glutamate receptor family. Nature 342:643–648Google Scholar
  10. Keinaenen K, Wisden W, Sommer B, Werner P, Herb A, Verdoorn TA, Sakmann B, Seeburg PH (1990) A family of AMPA-selective glutamate receptors. Science 249:556–560Google Scholar
  11. Komminoth P, Merk FB, Leav I, Wolfe HJ, Roth J (1992) Comparison of 35S- and digoxigenin-labelled RNA and oligonucleotide probes for in situ hybridization. Histochemistry 98:217–228Google Scholar
  12. Lyons GE, Schiaffino S, Sassoon D, Barton P, Buckingham M (1990) Developmental regulation of myosin gene expression in mouse cardiac muscle. J Cell Biol 111:2427–2436Google Scholar
  13. Maniatis T, Fritsch EF, Sambrook J (1982) Molecular cloning, a labatory manual. Cold Spring Harbour Laboratory, Cold Spring Harbor, NYGoogle Scholar
  14. Oh JD, Woolf NJ, Roghani A, Edwards RH, Butcher LL (1992) Cholinergic neurons in the rat central nervous system demonstrated by in situ hybridization of choline acetyltransferase mRNA. Neuroscience 47:807–822Google Scholar
  15. Ohtani H, Kuroiwa A, Obinata M, Ooshima A, Nagura H (1992) Identification of type I collagen-producing cells in human gastrointestinal carcinomas by non-radioactive in situ hybridization and immunoelectron microscopy. J Histochem Cytochem 40:1139–1146Google Scholar
  16. Raff MC, Miller RH, Noble M (1983) A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on culture medium. Nature 303:390–396Google Scholar
  17. Ross TS, Jefferson AB, Mitchell CA, Majerus PW (1991) Cloning and expression of human 75-kDa inositol polyphosphate-5-phosphatase. J Biol Chem 266:20283–20289Google Scholar
  18. Schaeren-Wiemers N, Schaefer C, Yancopoulos GD, Schwab ME (1991) A differential screening approach to isolate new oligodendrocyte-specific cDNA clones. Soc Neurosci Abstr 17:1149Google Scholar
  19. Springer JE, Robbins E, Gwag BJ, Lewis ME, Baldino F Jr (1991) Non-radioactive detection of nerve growth factor receptor (NGFR) mRNA in rat brain using in situ hybridization histochemistry. J Histochem Cytochem 39:231–234Google Scholar
  20. Verity AN, Levine MS, Campagnoni AT (1990) Gene expression in the jimpy mutant: evidence for fewer oligodendrocytes expressing myelin protein genes and impaired translocation of myelin basic protein mRNA. Dev Neurosci 12:359–372Google Scholar
  21. Wahle P, Beckh S (1992) A method of in situ hybridization combined with immunocytochemistry, histochemistry, and tract tracing to characterize the mRNA expressing cell types in heterogenous neuronal populations. J Neurosci Methods 41:153–166Google Scholar

Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Nicole Schaeren-Wiemers
    • 1
  • Andrea Gerfin-Moser
    • 1
  1. 1.Brain Research InstituteUniversity of ZürichZürichSwitzerland

Personalised recommendations