The in situ hybridization and immunocytochemistry techniques for characterization of cells expressing specific mRNAs in paraffin-embedded brains

In situ hybridization (ISH) represents a powerful and sensitive method for examining gene expression in individual cells in a manner analogous to immunocytochemistry (ICC) which is used to localize and identify cells containing a particular protein or labile antigens such as amino acid neurotransmitters. Considerable progress has been made both in molecular cloning which provides nucleotide probes and in the production of antibodies to specific antigens, especially those directed against small haptenes such as GABA and catecholamines. Consequently, specific tools have become available for ISH and ICC procedures in most cases. Several techniques including double ISH, double ICC and ISH coupled to ICC have been developed to characterize the phenotype of cells expressing neurotransmitters or specific neuroreceptors. This has been particularly well illustrated for neuropeptides (Young, III, W.S., Mezey, E. and Siegel, R.E. Vasopressin and oxytocin mRNAs in adrenalectomized and Brattleboro rats: analysis by quantitative in situ hybridization and histochemistry, Mol. Brain Res., 1 (1986) 231-241[17]) or dopamine receptors (Le Moine, C., Normand, E., Guitteny, A.F., Fouque, B., Teoule, R. and Bloch, B., Dopamine receptor gene expression by enkephalin neurons in rat forebrain, Proc. Natl. Acad. Sci. (USA), 87 (1990) 20-24 [8]; Le Moine, C., Normand, E. and Bloch, B., Phenotypical characterisation of the rat striatal neurons expressing the D1 dopamine receptor gene, Proc. Natl. Acad. Sci. (USA), 88 (1991) 4205-4209 [9]) but exclusively on frozen tissues. Combination of ISH and ICC allows the simultaneous detection of specific mRNAs and antigens in a same section or in adjacent sections. However, when ISH is coupled to ICC on a same section, a loss of signal in ISH is often observed (Siegel, R.E., Localization of neuronal mRNAs by hydridization histochemistry. In: P.M. Conn (Ed.), Methods in Neurosciences. Gene Probes. Vol. 1, Academic Press, New York, 1989, pp. 136-150 [13]) which may be a handicap when the rate of mRNA expression is low. Moreover, interferences between ISH coupled to ICC techniques may occur leading to artifactual signals. In the present protocol, in situ hybridization with a specific 35S-labeled riboprobe and immunocytochemistry with a polyclonal antibody are performed on adjacent sections of paraffin-embedded rodent brains. Paraffin embedding allows a better preservation of tissue morphology than freezing procedure. This is of particular importance since simultaneous detection of mRNAs and proteins in the same cell populations on adjacent sections requires conditions where (a) cell morphology is highly retained in a same manner on adjacent sections, and (b) reactivity of nucleic acids and antigens is preserved. We have used this technique to analyze the presence of serotonin 5-HT(1B) mRNA in GABAergic cell bodies (Cloëz-Tayarani, I., Wusher, N., Huerre, M. and Fillion, G., Cellular localization of 5-HT(1B) receptor mRNA in the rat olfactory tubercle: do GABAergic neurons express the 5-HT(1B) gene? Mol. Brain Res., 36 (1996) 337-342 [3]).