Summary
In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.
Article PDF
Similar content being viewed by others
Avoid common mistakes on your manuscript.
References
Agrawal S, Christodoulou C, Gait MJ (1986) Efficient methods for attaching non-radioactive labels to 5′ ends of synthetic oligodeoxyribonucleotides. Nucleic Acids Res 14:6227–6245
Allen YS, Adrian TE, Allen JM, Tatemoto K, Crow TJ, Bloom SR, Polak JM (1983) Neuropeptide Y distribution in the rat brain. Science 221:877–879
Baldino F Jr, Lewis ME (1989) Non-radioactive in situ hybridization histochemistry with digoxigenin-dUTP labelled oligonucleotides. Methods Neurosci 1:282–292
Burgunder J-M, Young WS 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
Chronwall BM, DiMaggio DA, Massari VJ, Pickel VM, Ruggiero DA, O'Donohue TL (1985) The anatomy of neuropeptide-Y-containing neurons of rat brain. Neuroscience 15:1159–1181
Dagerlind Å, Goldstein M, Hökfelt T (1990a) Most ganglion cells in the rat adrenal medulla are noradrenergic. Neuroreport 1:137–140
Dagerlind Å, Schalling M, Eneroth P, Goldstein M, Hökfelt T (1990b) Effect of reserpine on phenylethanolamine N-methyl-transferase mRNA levels in rat adrenal gland: role of steroids. Neurochem Int 17:343–356
De Jong ASH, Van Kessel-Van Vark M, Raap AK (1985) Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry. Histochem J 17:1119–1130
De Quidt ME, Emson PC (1986) Distribution of neuropeptide Y-like immunoreactivity in the rat central nervous system. II. Immunohistochemical analysis. Neuroscience 18:546–618
Fuxe K, HÖkfelt T, Said SI, Mutt V (1977) Vasoactive intestinal polypeptide and the nervous system: immunohistochemical evidence for localization in central and peripheral neurons, particularly intracortical neurons of the cerebral cortex. Neurosci Lett 5:241–246
Gehlert DR, Chronwall BM, Schafer MR, O'Donohue TL (1987) Localization of neuropeptide Y mRNA in rat and mouse brain by in situ hybridization. Synapse 1:25–31
Gendelman HE, Moench TR, Narayan O, Griffin DE, Clements JE (1985) A double labelling technique for performing immunocytochemistry and in situ hybridization in virus infected cell cultures and tissues. J Virol Methods 11:93–103
Gerfen CR, Young WS III (1988) Distribution of striatonigral and striatopallidal peptidergic neurons in both patch and matrix compartments: an in situ hybridization histochemistry and fluorescent retrograde tracing study. Brain Res 460:161–167
Grima B, Lamouroux A, Blanot F, Faucon-Biguet N, Mallet J (1985) Complete coding sequence of rat tyrosine hydroxylase mRNA. Proc Natl Acad Sci USA 82:617–621
Guitteny A-F, Böhlen P, Bloch B (1988) Analysis of vasopressin gene expression by in situ hybridization and immunohistochemistry in semi-thin sections. J Histochem Cytochem 36:1373–1378
Haralambidis J, Chai M, Treger GW (1987) Preparation of base-modified nucleotides suitable for non-radioactive label attachment and their incorporation into synthetic oligodeoxyribonucleotides. Nucleic Acids Res 15:4857–4876
Hökfelt T, Fuxe K, Goldstein M, Joh TH (1973) Immunohistochemical localization of three catecholamine synthesizing enzymes: aspects on methodology. Histochemie 33:231–254
Hökfelt T, Johansson O, Fuxe K, Goldstein M, Park D (1976) Immunohistochemical studies on the localization and distribution of monoamine neurons systems in the rat brain. I. Tyrosine hydroxylase in the mes- and diencephalon. Med Biol 54:427–453
Ichimiya Y, Emson PC, Christodulou C, Gait MJ, Ruth JL (1989) Simultaneous vizualization of vasopressin and oxytocin mRNA containing neurons in the hypothalamus using non-radioactive in situ hybridization histochemistry. J Neuroendocrinol 1:73–75
Jablonsky E, Moomaw EW, Tullis RH, Ruth J (1986) Preparation of oligonucleotide alkaline phosphatase conjugates and their use as hybridization probe. Nucleic Acids Res 14:6115–6128
Kiyama H, Emson PC, Ruth J (1990a) Distribution of tyrosine hydroxylase mRNA in the rat central nervous system visualized by alkaline phosphatase in situ hybridization histochemistry. Eur J Neurosci 2:512–524
Kiyama H, Emson PC, Ruth J, Morgan C (1990b) Sensitive non-radioactive in situ hybridization histochemistry: demonstration of tyrosine hydroxylase gene expression in rat brain and adrenal. Mol Brain Res 7:213–219
Kiyama H, Emson PC, Tohyama M (1990c) Recent progress in the use of the technique of non-radioactive in situ hybridization histochemistry: new tools for molecular neurobiology. Neurosci Res 9:1–21
Kiyama H, McGowan EM, Emson PC (1991) Co-expression of cholecystokinin mRNA and tyrosine hydroxylase mRNA in populations of rat substantia nigra cells; a study using a combined radioactive and non-radioactive in situ hybridization procedure. Mol Brain Res 9:87–93
Kuhar MJ (1981) Autoradiographic localization of drug and neurotransmitter receptors in the brain. Trends Neurosci 4:60–64
Larhammar D, Ericsson A, Persson H (1987) Structure and expression of rat neuropeptide Y gene. Proc Natl Acad Sci USA 84:2068–2072
Lorén I, Emson PC, Fahrenkrug J, Björklund A, Alumets J, Håkanson R, Sundler F (1979) Distribution of vasoactive intestinal polypeptide in the rat and mouse brain. Neuroscience 4:1953–1976
Nishizawa M, Hayakawa Y, Yanaihara N, Okamoto H (1985) Nucleotide sequence divergence and functional constraint in VIP precursor mRNA evolution between human and rat. FEBS Lett 183:55–59
Ozden S, Aubert C, Gonzalez-Dunia D, Brahic M (1990) Simultaneous in situ detection of two mRNAs in the same cell using riboprobes labelled with biotin and35S. J Histochem Cytochem 38:917–922
Pease PC (1962) Buffered formaldehyde as a killing agent and primary fixative for electron microscopy. Anat Rec 142:342
Ronnekleiv OK Naylor BR, Bond CT, Adleman JP (1989) Combined immunohistochemistry for gonadotropin-releasing hormone (GnRH) and pro-GnRH, and in situ hybridization for GnRH messenger ribonucleic acid in rat brain. Mol Endocrinol 3:363–371
Ruth J, Morgan C, Paska A (1985) Linker arm nucleotide analogs useful in oligonucleotide synthesis. DNA 4:93
Sambrook J, Fritsch EF, and Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbour Laboratory, Cold Spring Harbor, New York
Schalling M (1990) In situ hybridization studies on regulatory molecules in neural and endocrine tissue with special reference to expression of coexisting peptides. MD Thesis, Karolinska Institute, Stockholm. ISBN 91-628-0036-1
Schalling M, Hökfelt T, Wallace B, Goldstein M, Filer D, Yamin C, Schlesinger DH (1986) Tyrosine-3-hydroxylase in rat brain and adrenal medulla: hybridization histochemistry and immunohistochemistry combined with retrograde tracing. Proc Natl Acad Sci USA 83:6208–6212
Schalling M, Dagerlind Å, Brené S, Hallman H, Djurfeldt M, Persson H, Terenius L, Goldstein M, Schlesinger D, Hökfelt T (1988) Coexistence and gene expression of phenylethanolamine N-methyltransferase, tyrosine hydroxylase and neuropeptide tyrosine in the rat and bovine adrenal gland. Proc Natl Acad Sci USA 85:8306–8310
Schalling M, Dagerlind Å, Goldstein M, Ehrlich M, Greengard P, Hökfelt T (1990) Comparison of gene expression of the dopamin D-2 receptor and DARPP-32 in rat brain, pituitary and adrenal gland. Eur J Pharmacol 188:277–281
Seroogy K, Schalling M, Brené S, Dagerlind Å, Chai SY, Hökfelt T, Persson H, Brownstein M, Huan R, Dixon J, Filer D, Schlesinger D, Goldstein M (1989) Cholecystokinin and tyrosine hydroxylase messenger RNAs in neurons of rat mesencephalon: peptide/monoamine coexistence studies using in situ hybridization combined with immunohistochemistry. Exp Brain Res 74:149–162
Uhl GR (ed) (1986) In situ hybridization in brain. Plenum, New York London
Valentino KL, Eberwine JH, Barchas DJ (eds) (1987) In situ hybridization: application to neurobiology. Oxford University Press, Oxford New York
Van Noorden CJF, Jonges GN (1987) Quantification of the histochemical reaction for alkaline phosphatase activity using the indoxyl-tetranito BT method. Histochem J 19:94–102
Weisberg EP, Baruchin A, Stachowiak MK, Stricker E, Zigmond MJ, Kaplan BB (1989) Isolation of a rat adrenal cDNA clone encoding phenylethanolamine N-methyltransferase and cold-induced alterations in adrenal PNMT mRNA and protein. Mol Brain Res 6:159–166
Wilxox JN, Roberts JL, Chronwall BM, Bishop JF, O'Donohue T (1986) Localization of proopiomelanocortin mRNA in functional subsets of neurons defined by their axonal projections. J Neurosci Res 16:89–96
Young WS III (1989) Simultaneous use of digoxigenin- and radiolabelled oligodeoxyribonucleotide probes for hybridization histochemistry. Neuropeptides 13:271–275
Young WS III (1990) In situ hybridization histochemistry. In: Björklund A, Hökfelt T, Wouterlood FG, Pol AN van den (eds) Handbook of chemical neuroanatomy, vol 8. Elsevier, New York, pp 481–512
Young WS III, Hsu AC (1991) Observations on the simultaneous use of digoxigenin- and radiolabelled oligodeoxyribonucleotide probes for hybridization histochemistry. Neuropeptides 18:75
Young WS III, Mezey É, Siegel RE (1986a) Quantitative in situ hybridization histochemistry reveals increased levels of corticotropin-releasing factor mRNA after adrenalectomy in rats. Neurosci Lett 70:198–203
Young WS III, Mezey É, Siegel RE (1986b) Vasopressin and oxytocin mRNAs in adrenalectomized and Brattleboro rats: analysis by quantitative in situ hybridization histochemistry. Mol Brain Res 1:231–241
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Dagerlind, Å., Friberg, K., Bean, A.J. et al. Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry. Histochemistry 98, 39–49 (1992). https://doi.org/10.1007/BF00716936
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00716936