We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding preserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity.
The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electronmicroscopical level.
Bigbee JW, Kosek JC, Eng LF (1977) Effects of primary antiserum dilution on staining of “antigen-rich” tissues with the peroxidase antiperoxidase technique. J Histochem Cytochem 25: 443–447PubMedGoogle Scholar
Caldwell JD, Brooks PJ, Jirikowski GF, Barakat AS, Lund PK, Pedersen CA (1989) Estrogen alters oxytocin mRNA levels in the preoptic area. J Neuroendocrinol 1: 273–278Google Scholar
Ivell R, Richter D (1984) Structure and comparison of the oxytocin and vasopressin genes from rat. Proc Natl Acad Sci USA 81: 2006–2010PubMedCrossRefGoogle Scholar
Jäger G, Dörmer P (1981) Quantitation of the PAP-reaction on single cells by absorption cytophotometry. J Immunol Methods 44: 55–62PubMedCrossRefGoogle Scholar
Jirikowski GF, Ramalho-Ortigao JF, Seliger H (1988) In situ hybridization with complementary synthetic oligonucleotide and immunocytochemistry: a combination of methods to study transcription and secretion of oxytocin by hypothalamic neurons. Mol Cell Probes 2: 59–64PubMedCrossRefGoogle Scholar
Jirikowski GF, Ramalho-Ortigao JF, Lindl T, Seliger H (1989) Immunocytochemistry of 5 bromo-2′-deoxyuridine labelled oligonucleotide probes, a novel technique for in situ hybridization. Histochemistry 91: 51–53PubMedCrossRefGoogle Scholar
Kumar A, Tchen P, Roullet F, Cohen J (1988) Nonradioactive labelling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase. Anal Biochem 169: 376–382PubMedCrossRefGoogle Scholar
McCabe JJ, Morrell JI, Pfaff DW (1986) In situ hybridization to brain peptide m-RNAs. In: Uhl GR (ed) In situ hybridization in brain. Plenum Press, New York, pp 73–95Google Scholar
Naus CCG, Miller FD, Morrison JH, Bloom FE (1988) Immunohistochemical and In situ hybridization analysis of the development of the rat somatostatin-containing neocortical neuronal system. J Comp Neurol 269: 448–463PubMedCrossRefGoogle Scholar
Priestley JV, Haynes MA, Han VKM, Rethelyi M, Perl ER, Lund PK (1988) In situ hybridization using32P-labelled oligodeoxyribonucleotides for cellular localization of mRNA in neuronal and encrine tissue. Histochemistry 89: 467–478PubMedCrossRefGoogle Scholar
Ramalho-Ortigao JF, Jirikowski GF, Seliger H (1989) 5-Bromouridinylated oligonucleotide for hybridization analysis of DNA and RNA on membranes and in situ. Nucleosides Nucleotides 8: 805–813Google Scholar
Reisert I, Jirikowski GF, Pilgrim Ch, Oertel W, Marangos P (1982) The development of immunoreactivity for neuron-specific enolase (NSE) of preoptic and septal neurons in dissociated cultures. Neuroscience 7: 1317–1322PubMedCrossRefGoogle Scholar
Sinha ND, Biernat J, Köster H (1983) β-CyanoethylN,N-dialkyl amino/N-morpholinomonochloro phosphoamidites, new phosphitylating agents facilitating ease of deprotection and work-up of synthesized oligonucleotides. Tetrahedron Lett 24: 5843–5846CrossRefGoogle Scholar
Wallace RB (1986) Oligonucleotide probes for discrimination of RNAs that differ by a single nucleotide. In: Lerman LSt (ed) DNA probes: application in genetic and infectious disease and cancer. Cold Spring Harbor, New York, pp 127–131Google Scholar