ARTICLE

An In Situ Hybridization Histochemistry Technique Allowing Simultaneous Visualization by the Use of Confocal Microscopy of Three Cellular mRNA Species in Individual Neurons

Correspondence to: Michel Grino, Laboratoire de Neuroendocrinologie Expérimentale, INSERM U 297, UER de Médecine Nord, Blvd P. Dramard, 13916, Marseille Cedex 20, France.

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 -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. (J Histochem Cytochem 46:753–759, 1998)

Key Words: fuorescent in situ hybridization, radioactive in situ hybridization, multiple labelings, mRNA, laser confocal microscopy, paraventricular nucleus, arginine vasopressin, corticotropin-releasing factor, peptidylglycine -amidating, monooxygenase, digoxigenin, biotin

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