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An in situ hybridization histochemistry technique allowing simultaneous visualization by the use of confocal microscopy of three cellular mRNA species in individual neurons

An in situ hybridization histochemistry technique allowing simultaneous visualization by the use of confocal microscopy of three cellular mRNA species in individual neurons

Journal of Histochemistry and Cytochemistry: Official Journal of the Histochemistry Society 46(6): 753-759

ISSN/ISBN: 0022-1554

PMID: 9603787

DOI: 10.1177/002215549804600608

We present a specific and sensitive method for simultaneous detection of three mRNA species in individual neurons. The method relies on the use of riboprobes labeled with (35S)-UTP, digoxigenin-UTP, or biotin-UTP. The nonradioactive probes were sequentially revealed by incubation with anti-digoxigenin immunoglobulins or streptavidin conjugated to peroxidase, followed by the use of fluorochrome-labeled tyramides as peroxidase substrates. The radioactive probe was revealed by conventional autoradiography. There was no interaction among the different probes or the various detection systems. We demonstrate the use of this method by illustrating on laser scanning confocal microscopy the co-localization of the mRNAs coding for corticotropin-releasing factor (CRF), arginine vasopressin (AVP), or peptidylglycine alpha-amidating monooxygenase (PAM) in rat hypothalamic paraventricular nucleus (PVN) and its modulation by endogenous glucocorticoids. Our results suggest that this method could be used not only to study the regulation of the hypothalamo-pituitary-adrenal axis but also in various models in which mRNAs are present at low concentrations.

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Accession: 008146843

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