Localization of substance P mRNA in cholinergic cells of the rat laterodorsal tegmental nucleus:In situ hybridization histochemistry and immunocytochemistry

1. In situ hybridization histochemical techniques in combination with immunocytochemistry and acetylcholinesterase (AChE) histochemistry were used to study the colocalization of messenger RNA (mRNA) encoding the neuropeptide substance P (SP) in cholinergic cells of the laterodorsal tegmental nucleus (LDT) of the rat pontine brain stem. 2. Alternate serial sections were hybridized with a 48-base, 35S-labeled synthetic oligonucleotide probe encoding SP using in situ hybridization histochemistry and processed either histochemically for AChE or immunocytochemically for choline acetyltransferase (ChAT). 3. In addition, serial section analysis was used to demonstrate the correlation between SP and SP mRNA in the same cells of the LDT. 4. These studies reveal that the cholinergic neurons of the LDT synthesize SP.     

Identification of aneuploid cells in cytological specimens by combined in situ hybridization and immunocytochemistry

The purpose of our study was the application of non-isotopic in situ hybridization with chromosome-specific repetitive DNA probes for the determination of cytogenetically aberrant cells in routine cytological materials, such as cervical smears and breast tumour aspirates. Hyperdiploid cells in fine needle aspirates (FNA) of breast tumours could be visualized by in situ hybridization with a chromosome l-specific repetitive DNA probe. However, for the evaluation of a specific cell type in heterogeneous cell populations, i.e. cervical smears, a procedure combining immunocytochemistry and in situ hybridization can be required. Therefore, we developed a combination protocol using β-galactosidase/ ferri-ferrocyanide (blue-green) for immunocytochemistry and peroxidase/DAB (brown-black) for detection of the DNA probe. the described protocol enabled us to distinguish squamous epithelial cells within heterogeneous cell populations. By combining the chromosome 1 DNA probe with a specific cytokeratin marker it was possible to identify the chromosomal abnormal cells within cervical smears.     

Staining reaction for beta-galactosidase in immunocytochemistry and in situ hybridization.

beta-galactosidase, revealed by an indigo blue reaction product, represents a valid tracer in immunohistochemistry. Observations on the instability of the indigo precipitate led us to investigate this phenomenon. We conclude that avoiding of xylene, and mounting of the preparates in Histoclear (a xylene substitute) and Canada balsam (instead of synthetic resin mountants) yields a sharp and stable indigo precipitate. In addition, we propose a sensitive alternative staining procedure for beta-galactosidase, based on TNBT reduction and precipitation. These reactions can be used both in immunohistochemistry and in in situ hybridization.     

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.     

Glass slide models for immunocytochemistry and in situ hybridization

We have developed a model system based on the immobilization of microdroplets (0.1 l volumes) of antigens or of target (sense) oligonucleotides onto aminoalkylsilane-coated glass slides. Oligopeptide antigens need to be vapour-fixed in order to achieve efficient immobilization, while oligonucleotides do not require fixation. Protein antigens, exemplified by rabbit immunoglobulin, may be subjected to liquid fixation. The glass slide models are optically translucent and useful both for in situ hybridization and immunocytochemistry. The slides are compatible with detection systems of both light and fluorescence microscopy and permit measurement of staining intensities by microfluorimetry or computerized microdensitometry. The model systems can be used for comparisons of method sensitivities, for characterizing antibody and probe sensitivities and cross-reactivities, and as internal standards or quality controls for immunocytochemistry and in situ hybridization.     

Factors regulating the activity of striatal neurons: New perspectives fromin situ hybridization histochemistry

1. The basal ganglia contain a variety of putative peptide neurotransmitters. In situ hybridization allows changes in the levels of the mRNAs encoding these neuropeptides to be assessed at the cellular level of resolution. 2. Alterations in the activity of pathways within the basal ganglia of the rat produce distinct effects on the different neuropeptide mRNAs. 3. The evidence, where available, suggests that mRNA levels provide an index of peptide turnover. 4. This approach has consequently revealed much new information on the regulation of neuronal activity in the basal ganglia.     

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.     

Individual interphase chromosome domains revealed by in situ hybridization

Summary The position and arrangement of individual chromosomes in interphase nuclei were examined in mouse-human cell hybrids by in situ hybridization of biotinylated human DNA probes. Intense and even labeling of human chromosomes with little background was observed when polyethylene glycol and Tween-20 were included in hybridization solutions. Human interphase chromosomes were separated from each other in the nucleus, and were confined to well localized domains. Hybrid cells with a single human chromosome showed a reproducible position of this chromosome in the nucleus. Some chromosomes appeared to have a characteristic folding pattern in interphase. Optical section as well as electron microscopy of labeled regions revealed the presence of 0.2 m wide fibers in each interphase domain, as well as adjacent, locally extended 500 nm fibers. Such fibers are consistent with previously proposed structural models of interphase chromosomes.     

A simple method for coupling in situ hybridization and immunocytochemistry: application to the study of peptidergic neurons

We have devised a simple procedure for immunostaining of sections that have previously undergone autoradiographic visualization of mRNAs by in situ hybridization. Classical hybridocytochemistry techniques were performed first on cryostat sections of formaldehyde-fixed tissue. Standard methods were used for slide coating by emulsion dipping and for revelation, fixation, and coverslipping steps. The key to this method is the emulsion removal, or permeabilization, by a short trypsin incubation (0.2% for 20-30 sec) which facilitates the good access of antibodies used in a subsequent immunocytochemical technique to section epitopes. Usual immunofluorescence and immunoperoxidase procedures were successfully performed after this treatment. The immunoreactivity of several neuropeptides was well preserved after this procedure. In addition to its usefulness in our studies, this general method should be applicable to many other situations in which autoradiographic and immunocytochemical detections must be coupled.     

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.     

Direct eye visualization of Cy5 fluorescence for immunocytochemistry and in situ hybridization.

Cyanine 5.18 (or Cy5) is a fluorochrome emitting in the long-red/far-red range, usually regarded as unsuitable for direct observation by the human eye. We describe here the optimization of a direct visualization approach to Cy5 labeling, based on a standard fluorescence microscope with mercury light excitation and applicable to both immunocytochemistry and fluorescent in situ hybridization. Crucial factors were (a) an excitation path in the microscope not absorbing light in the orange-red range, up to 640 nm, (b) a 588-640-nm excitation filter range, distinctly below the excitation optimum for Cy5, (c) a 650-700-nm emission filter range, transmitting the low-wavelength portion of Cy5 emission, and (d) high-efficiency filter set components allowing a narrow gap between excitation and emission ranges without visible cross-talk of excitation light in the emission path.     

Combination of in situ hybridization and immunocytochemistry to detect messenger RNAs in identified CNS neurons and glia in tissue culture

We have developed a technique in which immunofluorescence is combined with in situ hybridization using cDNA and RNA probes to assess the expression and distribution of messenger RNAs (mRNA) by neurons and neuroglia in tissue cultures of the rat dentate gyrus. The probes used in this study include a cDNA probe for ribosomal RNA (rRNA) and an RNA probe (cRNA) for glial fibrillary acidic protein (GEAP), an intermediate filament protein subunit expressed by astrocytes in the central nervous system. Both ubiquitous (tubulin) and cell type-specific (MAP-2 and GEAP) antibodies were used to identify neurons and neuroglia in culture. Using this procedure, the mRNA for rRNA was found in the cell bodies and large processes of MAP-2-positive neurons and throughout the cytoplasm of GEAP-positive flat astrocytes. In process-bearing astrocytes, GEAP mRNA is concentrated in the cell body, although some hybridization also occurred in astrocyte cell processes. With this combined in situ hybridization-immunofluorescence technique, the expression and distribution of an mRNA can be examined in different immunocytochemically identified cell types under identical culture and hybridization conditions. It is also possible to determine if there is a differential subcellular distribution of an mRNA in a single cell and if the distribution of the mRNA reflects the distribution of the protein itself. Finally, this technique can be utilized to verify the specificity of probes for cell type-specific mRNAs and to determine appropriate hybridization conditions to produce a specific signal.     

Cytoplasmic localization of human repetitive DNA revealed by in situ hybridization

Previously, we showed that a human repetitive DNA sequence (Sau3A family) belonging to a satellite DNA is unstable and constantly excised from the chromosomes (R. Kiyama, H. Matsui, and M. Oishi, 1986, Proc. Natl. Acad. Sci. USA 83, 4665). The unusual property of the repetitive DNA, along with another repetitive DNA (Alu sequence), was further investigated by in situ hybridization in several different human cells including HeLa, bone marrow, and peripheral blood cells. We found that the excised repetitive DNA sequences are localized not only in nuclei, but also in cytoplasm. These results have confirmed the instability of these DNA sequences in the chromosomes and further suggest that the alpha satellite DNA and the Alu sequence which were excised from the chromosomes are released from nuclei to cytoplasm.     

The nucleolus organizers of diploid wheats revealed by in situ hybridization

Summary Labelled RNA, transcribed in vitro from wheat ribosomal DNA cloned in a bacterial plasmid, has been hybridised to metaphase chromosomes of five diploid wheats. Autoradiography of the chromosomes has provided unequivocal evidence that these genotypes possess two pairs of nucleolus organizer chromosomes. The diploid wheat accessions used possess widely differing numbers of ribosomal RNA genes.