Combination of non-radioactive and radioactive in situ hybridization with immunohistochemistry: a new method allowing the simultaneous detection of two mRNAs and one antigen in the same brain tissue section.

Abstract

We describe here a simple method for combining nonradioactive and radioactive in situ hybridization and immunohistochemistry on the same brain tissue section. This approach was first developed on the well-characterized hypothalamo-neurohypophyseal system, facilitating the optimization of the triple-labeling procedure and the verification of labeling specificity. We report the simultaneous detection of vasopressin (VP) mRNA with a digoxigenin-labeled oligonucleotide, oxytocin (OT) mRNA with a %-labeled oligonucleotide, and OT peptide in the same 12-pm ayostat section. This was performed on floating sections as follows: first, the two probes were hybridized simultaneously; second, the peptide was detected with an immunoperoxidase-DAB procedure; third, the digoxigenin-labeled probe was detected with an alkaline phosphatase-NBTIBCIF' tech- IntroductionThe phenotypic organization of the nervous system is extremely complex. Many informative molecules, such as classical neurotransmitters and neuropeptides, have been demonstrated to be coexpressed in neuronal populations (15). For example, several peptides, as well as tyrosine hydroxylase, have been detected in the hypothalamo-neurohypophyseal system (6,24). Histochemical techniques, such as immunohistochemistry and in situ hybridization, became powerful tools for study of such phenotypic structure. However, single immuno-or hybridohistochemical labeling does not allow the precise analysis of the coexpression phenomena that frequently occur in neuronal cells. Such analyses can be made by performing multiple labelings involving immunohistochemistry and/or in situ hybridization in the same tissue sample. nique; and finally, the 35S-labeled probe was detected by histological autoradiography. We also demonstrate that this approach is suitable for the simultaneous detection of tyrosine hydroxylase and two less abundant mRNAs, vasoactive intestinal peptide and vasopressin "As, in the suprachiasmatic nucleus. The combination of the three techniques did not significantly diminish their specificity or sensitivity. In conclusion, this new method, permitting the simultaneous detection of three different products of gene expression in the same section, could be useful for further analysis of the phenotypic organization and its plasticity in endocrine or neural tissues. In recent years the sensitivity of in situ hybridization techniques has increased, allowing the detection of mRNAs encoding various informative molecules, using both radioactive and non-radioactive probes (4,12,41). Moreover, double labeling involving double in situ hybridization of two different probes has been successfully performed. This was done, for example, by combining the use of a radiolabeled probe and a biotin-(28,29,33), a digoxigenin-(42), or an alkaline phosphatase-labeled probe (21). Double nonradioactive in situ hybridization has also been developed by use of fluorescent (11) or enzymatic (17) markers. In addition, in situ hybridization histochemistry has been coupled with immunohistochemistry in sever...

Expression of the pituitary transcription factor GHF-1/PIT-1 in cell types of the adult porcine adenohypophysis.

“…In our case, the different origin of the antisera used (i.e., sheep polyclonal antiserum for digoxigenin-labeled probe and rabbit polyclonal antisera for pituitary hormones) allowed the simultaneous incubation of both antisera, therefore reducing the procedure time. As previously reported (Trembleau et al, 1993;Ichiyama et al, 1989). our results confirm that there is no interaction between the peroxidase and alkaline phosphatase detection systems.…”

“…Indeed, this transcription factor appears to be highly conserved in vertebrates (Martinez-Barberi et al, 1994;Elsholtz et al, 1992). Double-labeling techniques are useful and precise methods for determining co-localization of mRNA and proteins (Trembleau et al, 1993). In our case, the different origin of the antisera used (i.e., sheep polyclonal antiserum for digoxigenin-labeled probe and rabbit polyclonal antisera for pituitary hormones) allowed the simultaneous incubation of both antisera, therefore reducing the procedure time.…”

Triple-labeling method combining immunocytochemistry and in situ hybridization histochemistry: demonstration of overlap between Fos-immunoreactive and galanin mRNA-expressing subpopulations of luteinizing hormone-releasing hormone neurons in female rats.

“…First, to successfully combine ICC and ISHH, tissues must be fixed sufficiently to retain proteins of interest, but not so heavily fixed that the mRNA is not readily accessible to probes. We found, in agreement with previous studies (31,32), that perfusion-fixation with 4% paraformaldehyde in PBS satisfies these criteria. Penetration of the probe did not pose a problem, even when we used a cRNA probe that was approximately seven or eight times longer than synthetic deoxynucleotidyl probes used in previous studies of perfused tissues (31,32).…”

Mononuclear Cell-Infiltrate Inhibition by Blocking Macrophage-Derived Chemokine Results in Attenuation of Developing Crescentic Glomerulonephritis

“…After dehydration, emulsion-dipped slides were exposed for 3 weeks at 4°C then, developed, fixed, and counterstained with hematoxylin. 48,49 Results…”