Simultaneous detection of two messenger RNAs in the central nervous system: a simple two-step in situ hybridization procedure using a combination of radioactive and non-radioactive probes

We present here a method enabling the simultaneous detection of two messenger RNAs in tissue sections by use of a two-step in situ hybridization procedure. Tissue sections were hybridized with a radioactive probe and coated with emulsion. The emulsion was processed for development, fixed, and a second hybridization was performed through the emulsion with a biotinylated probe subsequently revealed with streptavidin-alkaline phosphatase. This procedure allows the detection of two mRNAs without loss of signal, removal of the emulsion, or spurious reaction. The simultaneous detection of oxytocin and vasopressin mRNAs in the hypothalamus, and of dopamine receptor and neuropeptide mRNAs in the striatum, demonstrated the efficiency of the procedure. Such a two-step procedure provides a simple and flexible way to make possible comparative analysis of the localization of two mRNAs within the same tissue section.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry

Summary Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry

Summary In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using³⁵S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using³⁵S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (<1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Detection of gastrin and its messenger RNA in Zollinger-Ellison tumors by non-radioactive in situ hybridization and immunocytochemistry.

Gastrin immunocytochemistry and non-radioactive in situ hybridization, using biotinylated oligonucleotide probes, for gastrin mRNA have been used for studying a retrospective material of six gastrin-producing (Zollinger-Ellison) tumors. Hybridization results for gastrin mRNA were positive in all six, while gastrin immunoreactivity could be detected in five tumors. In one of the patients, different areas of the same tumor displayed differences in immunoreactivity to gastrin, but were uniformly hybridization positive. Weak hybridization signals were detected in liver metastases from a necropsy case, while the gastrin immunostaining was more pronounced. The results show that non-radioactive hybridization methods are applicable to routine clinical specimens stored for as long as 16 years and that in situ hybridization may be a useful complement to immunocytochemical diagnosis, particularly in cases where high synthesis and little storage of hormonal products occur.     

Sensitive mRNA detection using unfixed tissue: combined radioactive and non-radioactive in situ hybridization histochemistry.

In the present study some experimental parameters for in situ hybridization histochemistry (ISHH) have been analysed using 35S-labelled and alkaline phosphatase-conjugated probes, in order to develop a reproducible double-labelling procedure. We have compared the total exclusion of tissue fixation with tissue sections fixed by immersion in formalin. In addition, the effect of dithiothreitol was assessed both when combining radiolabelled and non-radioactive probes on a single tissue section and when the probes were used separately. Hybridization of unfixed tissue resulted in stronger specific labelling and lower background both for radiolabelled and alkaline phosphatase-conjugated probes. No loss in tissue preservation was seen at the light microscopic level after hybridization of unfixed tissue. High concentrations (200 mM) of dithiothreitol strongly suppressed background when using 35S-labelled probes, whereas in the non-radioactive procedure, alkaline phosphatase labelling could only be achieved with very low dithiothreitol concentrations (less than 1 mM). This incompatibility led to a protocol using unfixed tissue sections and a sequential hybridization procedure, with the radiolabelled probe and high concentrations of dithiothreitol in the first step and the alkaline phosphatase-conjugated probe without dithiothreitol in the second step.     

Electron microscopic detection of RNA sequences by non-radioactive in situ hybridization in the mollusk Lymnaea stagnalis.

The subcellular localization of mRNA sequences encoding neuropeptides in neuropeptidergic cells of the pond snail Lymnaea stagnalis was investigated at the electron microscopic (EM) level by non-radioactive in situ hybridization. Various classes of probes specific for 28S rRNA and for the ovulation hormone (caudodorsal cell hormone; CDCH) mRNA were labeled with biotin or digoxigenin and were detected after hybridization with gold-labeled antibodies. Hybridizations were performed on ultra-thin sections of both Lowicryl-embedded and frozen cerebral ganglia, and a comparison demonstrated that most intense hybridization signals with an acceptable preservation of morphology were obtained with ultra-thin cryosections. Addition of 0.1% glutaraldehyde to the formaldehyde fixative improved the morphology, but on Lowicryl sections this added fixative resulted in a decrease of label intensity. A variety of probes, including plasmids, PCR products, and oligonucleotides, were used and all provided good results, although the use of oligonucleotides on Lowicryl sections resulted in decreased gold labeling. The gold particles were found mainly associated with rough endoplasmic reticulum (RER) but were also observed in lysosomal structures. Finally, the in situ hybridization method presented in this study proved to be compatible with the immunocytochemical detection of the caudodorsal cell hormone, as demonstrated by double labeling experiments.     

Bi-color detection of two target DNAs by non-radioactive in situ hybridization

Summary A non-radioactive in situ hybridization technique is described which allows the simultaneous detection of different DNA sequences. To demonstrate the feasibility of the proccdure, metaphases and interphase nuclei of a human-mouse somatic cell hybrid were simultaneously hybridized with mercurated total human DNA and a biotinylated mouse satellite DNA probe. After the hybridization, the probes were detected immunocytochemically using two different and independent affinity systems. By this approach we visualized the two DNA target sequences in metaphase chromosomes and in interphase nuclei with FITC and TRITC fluorescence, or blue (alkaline phosphatase) and brown (peroxidase) precipitated enzyme products. This method not only allows detection of intact chromosomes but also the visualization of rearrangements between parts of human and mouse chromosomes. Furthermore, the technique demonstrates the high topological resolution of nonradioactive in situ hybridizations.This investigation was supported in part by FUNGO, Foundation of Medical Scientific Research in The Netherlands (grant nr 13-54-21)     

Assessment of decalcifying protocols for detection of specific RNA by non-radioactive in situ hybridization in calcified tissues

For the best performance of in situ analysis of specific RNA expression in calcified tissues, it is necessary to choose an appropriate protocol to decalcify the tissues. We evaluated the usefulness of various acid-based decalcifying reagents with reference to 28 S rRNA staining by in situ hybridization using a thymine-thymine dimerized oligonucleotide probe. The reagents evaluated were 10% nitric acid, 10% HCl, 5% formic acid, 5% trichloroacetic acid, Morse’s solution, Plank-Rychlo’s solution, and K-CX solution, all of which are commonly used to decalcify tissues, and their effects on retention of morphology and RNA were compared with EDTA-based solutions. When normal mouse mandible was used as a model tissue, well-preserved morphology of ameloblasts was obtained from sections decalcified with Morse’s solution, 10% HCl, Plank-Rychlo’s solution, and K-CX solution, and best retention of 28 S rRNA was obtained with 5% formic acid and Morse’s solution. We recommend Morse’s solution to decalcify tissues to be processed for the rapid analysis of specific RNA expression. Indeed, we detected specific mRNAs strongly in sections treated with Morse’s solution, and quantitative analysis showed that the ratio of signal intensities of 28 S rRNA and the specific mRNAs correlated with each other depending on decalcifying solutions. Accepted: 3 January 2000     

Ultrastructural detection of messenger RNA coding for growth hormone in the rat anterior pituitary by in situ hybridization

Ultrastructural detection of the messenger RNA coding for growth hormone in rat pituitary gland could be obtained by association of in situ hybridization and cryoultramicrotomy. Messenger RNAs were localized in the anterior pituitary gland. Silver grain densities observed in autoradiograms after in situ hybridization were dependent to incubation period and to fixation. It was necessary to determine a compromise between ultrastructural aspect and silver grain densities. Messenger RNAs were detected in somatotropic cells, identified by ultrastructural characteristics, in both the nucleus (euchromatin and nuclear membrane) and cytoplasm, in vicinity to endoplasmic reticulum.     

Quantitative detection of messenger RNA by solution hybridization and enzyme immunoassay

A novel nucleic acid detection technique is described for the quantitative measurement of eukaryotic mRNA in biological samples. The procedure involves two steps: a hybridization reaction in solution with a biotinylated cDNA probe, and a conventional enzyme immunoassay that uses a monoclonal antibody for DNA.RNA hybrids to detect the specific mRNA.cDNA complexes. The method has comparable sensitivity to 32P-based methods and yields results that are quantitative and highly reproducible. Furthermore, the test can be performed using unfractionated cytoplasm without the need for extraction with organic solvents. This technique provides a rapid and quantitative method for studying changes in cellular mRNA levels, and it is suitable for testing large numbers of samples.     

Distribution of the beta-2 adrenergic receptor messenger RNA in the rat brain by in situ hybridization histochemistry: Effects of chronic reserpine treatment

We studied the distribution of the rat brain beta-2 adrenergic receptor (AR) mRNA, and the effects of monoamine depletions by chronic reserpine treatment using in situ hybridization histochemistry. In the control group, high level signals of beta-2 AR mRNA were observed in the parietal, frontal and piriform cortices, the medial septal nuclei, the olfactory tubercle, and the midbrain. Moderate signals were found in the striatum, the retrosplenial cortex, the hippocampus, and the thalamic nuclei. After chronic reserpine treatment, beta-2 AR mRNA levels were increased in many brain regions. The large increases were seen in the hippocampus, all thalamic nuclei, the amygdaloid nuclei, and the midbrain, followed by the striatum and the occipital cortex. The receptor up-regulation resulting from chronic monoamine depletion may be due to these increases in beta-2 AR mRNA, indicating that this up-regulation may be caused by increased receptor production rather than decreased receptor degradation.     

Localization of urokinase-type plasminogen activator messenger RNA in the normal mouse by in situ hybridization

The histological distribution of urokinase-type plasminogen activator (u-PA) mRNA was analyzed in normal mouse tissue by in situ hybridization with anti-sense RNA transcribed from three different subclones of a mouse u-PA cDNA. Hybridization signal was found over a distinct fibroblast-like cell in the lamina propria of the gastrointestinal tract, over proximal, distal, and collecting tubules of the kidney, and over the epithelium of the bladder, ductus deferens, and epididymis. No hybridization signal was found over cells of the lung, pancreas, liver, adrenal, pituitary, cerebrum, hypothalamus, cerebellum, sciatic nerve, and striated muscle, nor over endothelial cells in any tissue investigated. The lack of u-PA mRNA in lung tissue was confirmed by Northern analysis and is in contrast to the high amounts of u-PA protein found in this tissue.     

Cell-specific detection of microRNA expression during cardiomyogenesis by combined in situ hybridization and immunohistochemistry

MicroRNAs (miRNAs) regulate gene expression by mediating translational repression or mRNA degradation of their targets, and several miRNAs control developmental decisions through embryogenesis. In the developing heart, miRNA targets comprise key players mediating cardiac lineage determination. However, although several miRNAs have been identified as differentially regulated during cardiac development and disease, their distinct cell-specific localization remains largely undetermined, likely owing to a lack of adequate methods. We therefore report the development of a markedly improved approach combining fluorescence-based miRNA-in situ hybridization (miRNA-ISH) with immunohistochemistry (IHC). We have applied this protocol to differentiating embryoid bodies (EBs) as well as embryonic and adult mouse hearts, to detect miRNAs that were upregulated during EB cardiomyogenesis, as determined by array-based miRNA expression profiling. In this manner, we found specific co-localization of miR-1 to myosin positive cells (cardiomyocytes) of EBs, developing and mature hearts. In contrast, miR-125b and -199a did not localize to cardiomyocytes, as previously suggested for miR-199a, but were rather expressed in connective tissue cells of the heart. More specifically, by co-staining with α-smooth muscle actin (α-SMA) and collagen-I, we found that miR-125b and -199a localize to perivascular α-SMA⁻ stromal cells. Our approach thus proved valid for determining cell-specific localization of miRNAs, and the findings we present highlight the importance of determining exact cell-specific localization of miRNAs by sequential miRNA-ISH and IHC in studies aiming at understanding the role of miRNAs and their targets. This approach will hopefully aid in identifying relevant miRNA targets of both the heart and other organs.     

Simultaneous application of in situ DNA hybridization and immunohistochemistry on one tissue section

Summary Combined application of a non-radioactivein situ DNA hybridization procedure and the immunoperoxidase technique on one tissue section is described. Of six potential protocols, only one proved to be successful. First, the immunohistochemical procedure including visualization of enzyme activity is performed; thein situ DNA hybridization protocol is then applied. Using this protocol, several antigens, detected with monoclonal antibodies, and target DNAs, detected by using biotinylated human cytomegalovirus or human papilloma virus type 16 DNA probes, could be distinguished by their peroxidase activity (brown precipitate) and alkaline phosphatase activity (purple—blue precipitate) respectively. The method allows immunophenotyping of virus-infected cells as well as simultaneous visualization of two viral parameters. This technique has important implications for research and diagnostic purposes.     

Neuroanatomical localization of cells containing gonadotropin-releasing hormone messenger ribonucleic acid in the primate brain by in situ hybridization histochemistry

Using in situ hybridization histochemistry, we have mapped the anatomic localization of perikarya containing mRNA that codes for GnRH and GnRH-associated protein (GAP) in the forebrain of four male macaques, Macaca fascicularis. DNA oligomers, with sequences complementary to either the GnRH or the GAP portion of the mRNA sequence, were synthesized and hybridized to paraformaldehyde fixed, coronal sections of the basal forebrain and hypothalamus. GnRH mRNA was found in the same population of cells as those containing GAP mRNA. GnRH/GAP mRNA-containing cell bodies were observed consistently in the medial septal nucleus, the diagonal band of Broca, the medial preoptic area, supraoptic nucleus, and ventromedial-infundibular region. We detected the presence of GnRH mRNA and GAP mRNA within the same neuroanatomic regions previously shown to include perikarya containing immunoreactive GnRH. The ventromedial-infundibular region and the medial preoptic region contained the greatest number of GnRH/GAP mRNA-containing perikarya (37.0% and 22.5%, respectively). The diagonal band contained 21.0% and the supraoptic nucleus 13.0% of the cells, while the medial septum contained the fewest number (6.7%). This study demonstrates the feasibility of using in situ hybridization as a strategy to study the developmental and steroidal regulation of GnRH gene expression in the nonhuman primate.     

Detection of the messenger RNA coding for preproenkephalin A in bovine adrenal by in situ hybridization

The messenger RNA (mRNA) coding for the adrenal precursor of enkephalins (preproenkephalin-A) has been detected in bovine adrenal medulla cells using in situ hybridization with 32P-labelled preproenkephalin A (PPA) complementary DNA. In formaldehyde- and Carnoy-fixed tissue sections, an intense elective labelling restricted to the cells located at the periphery of the adrenal medulla can be detected after hybridization procedure, using X-ray film and classical autoradiographic procedure. Adequate controls show that this labelling is obtained only using PPA complementary DNA, inserted or not in its vector. Distribution of PPA mRNA appears identical to that of its immunoreactive end products, namely Met-enkephalin and BAM22 peptide, detected by immunohistochemistry. Norepinephrine, detectable using monoamine histofluorescence, appears restricted to the cells of the center of the gland unlabelled for PPA mRNA and its end-products. Cultured bovine adrenomedullary cells that exhibited enkephalin immunoreactivity also contain PPA mRNA located in their cytoplasm.