Histochemistry

, Volume 89, Issue 5, pp 467–479 | Cite as

In situ hybridization using 32P labelled oligodeoxyribonucleotides for the cellular localisation of mRNA in neuronal and endocrine tissue

An analysis of procedural variables
  • J. V. Priestley
  • M. A. Hynes
  • V. K. M. Han
  • M. Réthelyi
  • E. R. Perl
  • P. K. Lund
Article

Summary

Methodological variables for in situ hybridization using 32P labelled oligodeoxyribonucleotides (oligomers) have been examined. Four different oligomers directed against proglucagon messenger RNA (mRNA) and two different oligomers against prosomatostatin mRNA have been used. Specific hybridization was obtained in adult rat brain, stomach and pancreas and in neonatal rat ileum. Tissue was perfusion fixed with 4% paraformaldehyde 0.2% glutaraldehyde and hybridization was carried out in 50% formamide for 72 h at 42° C. Using hybridization conditions of lower stringency (33% formamide) labelling was also obtained in guinea pig tissue. Other variables which affected hybridization signal intensity were the inclusion of a prehybridization dehydration stage, the probe concentration, the inclusion of ammonium acetate in the posthybridization dehydrating ethanols and in the autoradiographic emulsion, and the exposure time. The localisation of proglucagon mRNA in rat pancreas using a 20mer was used as a model tissue for testing these methodological variables and the results were found generally also to apply to the other probes and tissues tested. The methods described provide single cell resolution and show that 32P labelled oligomers may be used to localise neuropeptide and endocrine mRNAs in different types of tissue and in different mammalian species.

Keywords

Oligomer Formamide Ammonium Acetate Methodological Variable Specific Hybridization 

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References

  1. Angerer LM, Angerer RC (1981) Detection of poly A+ RNA in sea urchin eggs and embryos by quantitative in situ hybridisation. Nucleic Acids Res 9:2819–2840Google Scholar
  2. Arentzen R, Baldino F Jr, Davis LG, Higgins GA, Lin Y, Manning RW, Wolfsen B (1985) In situ hybridization of putative somatostatin mRNA within hypothalamus of the rat using synthetic oligonucleotide probes. J Cell Biochem 27:415–422Google Scholar
  3. Herod A, Hartman BK, Pujol JF (1981) Importance of fixation in immunohistochemistry: Use of formaldehyde solutions at variable pH for the localization of tyrosine hydroxylase. J Histochem Cytochem 29:844–850Google Scholar
  4. Bloch B, Robert JM, Baird A, Gubler U, Reymond C, Bohlen P, Le Guellec D, Bloom FE (1984) Detection of the messenger RNA coding for preproenkephalin A in bovine adrenal by in situ hybridization. Regul Pept 8:345–354Google Scholar
  5. Bloch B, Le Guellec D, De Keyser Y (1985) Detection of the messenger RNA's coding for the opioid peptide precursors in pituitary and adrenal by ‘in situ’ hybridization: Study in several mammal species. Neurosci Lett 53:141–148Google Scholar
  6. Chrigwin JM, Przybyla AE, MacDonald J, Rutter WJ (1979) Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294–5299Google Scholar
  7. Coghlan JP, Penschow JD, Hudson PJ, Niall HD (1984) Hybridization histochemistry: Use of recombinant DNA for tissue localizations of specific mRNA populations. Clin Hypertens [A] (1 and 2):63–78Google Scholar
  8. Connaughton M, Priestley JV, Sofroniew MV, Eckenstein F, Cuello AC (1986) Inputs to motoneurones in the hypoglossal nucleus of the rat: Light and electron microscopic immunocytochemistry for choline acetyltransferase, substance P and enkephalins using monoclonal antibodies. Neuroscience 17:205–224Google Scholar
  9. Gee CE, Roberts JL (1983) Laboratory methods. In situ hybridization histochemistry: A technique for the study of gene expression in single cells. DNA 2:157–163Google Scholar
  10. Gee CE, Chen CC, Roberts JL, Thompson R, Watson SJ (1983) Identification of proopiomelanocortin neurones in rat hypothalamus by ‘in situ’ cDNA-mRNA hybridization. Nature 306:374–376Google Scholar
  11. Goodman RH, Jacobs JW, Dee PC, Habener JF (1981) Somatostatin — 28 encoded in a cloned cDNA obtained from a rat medullary thyroid carcinoma. J Biol Chem 257:1156–1159Google Scholar
  12. Han VKM, Hynes MA, Jin C, Towle AC, Lauder JM, Lund PK (1986) Cellular localization of proglucagon/glucagon-like peptide I messenger RNAs in rat brain. J Neurosci Res 16:97–107Google Scholar
  13. Heinrich G, Gros P, Lund PK, Bentley RC, Habener JF (1984) Pre-proglucagon messenger ribonucleic acid: Nucleotide and encoded amino acid sequences of the rat pancreatic complementary deoxyribonucleic acid. Endocrinology 115:2176–2181Google Scholar
  14. Hoefler H, Childers M, Montminy MR, Lechan RM, Goodman RH, Wolfe HJ (1986) In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes. Histochem J 18:597–604Google Scholar
  15. Hökfelt T, Efendic S, Hellerström C, Johansson O, Luft R, Arimura A (1975) Cellular localization of somatostatin in endocrine-like cells and neurons of the rat with special reference to the A1-cells of the pancreatic islets and to hypothalamus. Acta Endocrinol (Copenh) 80 (Suppl 200):3–41Google Scholar
  16. Hudson P, Penschow J, Shine J, Ryan G, Niall H, Coghlan J (1981) Hybridization histochemistry: Use of recombinant DNA as a ‘homing probe’ for tissue localisation of specific mRNA populations. Endocrinology 108:353–356Google Scholar
  17. Jin C, Hynes MA, Lund PK (1987) Ontogeny of proglucagon mRNA in rat intestine. Endocr Soc Abstracts: 446Google Scholar
  18. Johansson O, Hökfelt T, Elde RP (1984) Immunohistochemical distribution of somatostatin-like immunoreactivity in the central nervous system of the adult rat. Neuroscience 13:265–339Google Scholar
  19. Larsson LI, Moody AJ (1980) Glicentin and gastric inhibitory polypeptide immunoreactivity in endocrine cells of the gut and pancreas. J Histochem Cytochem 28:925–933Google Scholar
  20. Larsson LI, Goltermann N, de Magistris L, Rehfeld JF, Schwartz TW (1979) Stomatostatin cell processes as pathways for paracrine secretion. Science 205:1393–1395Google Scholar
  21. Lewis ME, Sherman TG, Watson SJ (1985) In situ hybridization histochemistry with synthetic oligonucleotides: Strategies and methods. Peptides 6:75–87Google Scholar
  22. Lund PK, Moats-Staats BM, Simons JG, Hoyt E, D'Ercole AJ, Martin F, Van Wyk JJ (1985) Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor. J Biol Chem 260:7609–7613Google Scholar
  23. Lund PK, Perl ER, Priestley JV (1986) Localization of somatostatin mRNA in rat brain by in situ hybridization. J Physiol (London) 372:18PGoogle Scholar
  24. McAllister LB, Scheller RH, Kandel ER, Axel R (1983) In situ hybridization to study the origin and fate of identified neurons. Science 222:800–808Google Scholar
  25. Montminy MR, Goodman RH, Horovutch SJ, Habener JF (1984) Primary structure of the gene encoding somatostatin. Proc Natl Acad Sci USA 81:3337–3340Google Scholar
  26. Nawa H, Kotani H, Nakanishi S (1984) Tissue-specific generation of two preprotachykinin mRNAs from one gene by alternative RNA splicing. Nature 312:729–734Google Scholar
  27. Nojiri H, Sato M, Urano A (1985) In situ hybridization of the vasopressin mRNA in the rat hypothalamus by use of a synthetic oligonucleotide probe. Neurosci Lett 58:101–105Google Scholar
  28. Pool CW, Buijs RM, Swaab DF, Boer GS, Van Leeuwen FW (1983) On the way to specific immunocytochemical localization. In: Cuello AC (ed) Immunohistochemistry. John Wiley, London New York, pp 1–46Google Scholar
  29. Priestley JV (1987) Immunocytochemical techniques for the localisation of neurochemically characterised nerve pathways. In: Bachelard H, Turner A (eds) Neurochemistry, a practical approach. IRL Press, Oxford, pp 65–112Google Scholar
  30. Réthelyi M, McGehee D, Lund PK (1986) Neuronal localization of cholecystokinin mRNAs in rat and guinea pig brain. Soc Neurosci Abstr 12:1041Google Scholar
  31. Rosenfeld MG, Mermod JJ, Amara S, Swanson LW, Sawchenko PE, Rivier J, Vale WW, Evans RM (1983) Production of a novel neuropeptide encoded by the calcitonin gene via tissue-specific RNA processing. Nature 304:129–135Google Scholar
  32. Schultzberg M, Hökfelt T, Nilsson G, Terenius L, Rehfeld JF, Brown M, Elde R, Goldstein M, Said S (1980) Distribution of peptide- and catecholamine-containing neurons in the gastrointestinal tract of rat and guinea pig: immunohistochemical studies with antisera to substance P, vasoactive intestinal polypeptide, enkephalins, somatostatin, gastrin/cholecystokinin, neurotensin and dopamine β-hydroxylase. Neuroscience 5:689–744Google Scholar
  33. Shen LP, Pictet RL, Rutter WJ (1982) Human somatostatin I: Sequence of the cDNA. Proc Natl Acad Sci USA 79:4575–4579Google Scholar
  34. Siegel RE, Young WS (1986) Detection of preprocholecystokinin and preproenkephalin A mRNA in rat brain by hybridization histochemistry using complementary RNA probes. Neuropeptides 6:573–580Google Scholar
  35. Thomas PS (1980) Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci USA 77:5201–5205Google Scholar
  36. Towle AC, Lauder JM, Joh TH (1984) Optimization of tyrosine hydroxylase immunocytochemistry in paraffin sections using pretreatment with proteolytic enzymes. J Histochem Cytochem 32:766–770Google Scholar
  37. Uhl GR, Sasek CA (1986) Somatostatin mRNA: Regional variation in hybridization densities in individual neurons. J Neurosci 6:3258–3264Google Scholar
  38. Uhl GR, Zingg HH, Habener JF (1985) Vasopressin mRNA in situ hybridization: Localization and regulation studied with oligonucleotide cDNA probes in normal and Brattleboro rat hypothalamus. Proc Natl Acad Sci USA 82:5555–5559Google Scholar
  39. Vaillant CR, Lund PK (1986) Distribution of glucagon-like peptide I in canine and feline pancreas and gastrointestinal tract. J Histochem Cytochem 34:1117–1121Google Scholar
  40. Varndell IM, Bishop A, Sikri K, Uttenthal L, Bloom SR, Polak JM (1985) Localization of glucagon-like peptide immunoreactants in human gut and pancreas using light and electron microscopic immunocytochemistry. J Histochem Cytochem 33:1080–1086Google Scholar
  41. Wallace RB, Schaffer J, Murphy RF, Bonner J, Hirose T, Itakura K (1979) Hybridization of synthetic oligonucleotide to φ x DNA: The effect of single base pair mismatch. Nucleic Acids Res 6:3543–3557Google Scholar
  42. Wolfson B, Manning RW, Davis LG, Arentzen R, Baldino F Jr (1985) Co-localization of corticotropin releasing factor and vasopressin mRNA in neurones after adrenalectomy. Nature 315:59–61Google Scholar
  43. Young WS, Bonner TI, Brann MR (1986) Mesencephalic dopamine neurons regulate the expression of neuropeptide mRNAs in the rat forebrain. Proc Natl Acad Sci USA 83:9827–9831Google Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • J. V. Priestley
    • 1
    • 2
  • M. A. Hynes
    • 1
  • V. K. M. Han
    • 1
  • M. Réthelyi
    • 1
    • 3
  • E. R. Perl
    • 1
  • P. K. Lund
    • 1
  1. 1.Department of PhysiologyUniversity of North Carolina at Chapel HillChapel HillUSA
  2. 2.Departments of Physiology and BiochemistryUnited Medical and Dental Schools, St. Thomas's CampusLondonEngland
  3. 3.2nd Department of AnatomySemmelweis University, Medical SchoolBudapestHungary

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