In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.     

In situ hybridization: use of 35S-labeled probes on paraffin tissue sections

The following protocol is for radioactive in situ hybridization detection of RNA using paraffin-embedded tissue sections on glass microscope slides. Steps taken to inhibit RNase activity such as diethyl pyrocarbonate (DEPC) treatment of solutions and baked glassware are unnecessary. The tissue is fixed using 4% paraformaldehyde, hybridized with (35)S-labeled RNA probes, and exposed to nuclear-track emulsion. The entire procedure takes 2-3 days prior to autoradiography. The time required for autoradiography is variable with an average time of 10 days. Parameters that affect the length of the autoradiography include: (1) number of copies of mRNA in the tissue, (2) incorporation of label into the probe, and (3) amount of background signal. Additional steps involved in the autoradiography process, including development of the emulsion, cleaning of the microscope slides, counterstaining of the tissue, and mounting coverslips on the microscope slides, are discussed. In addition, a general guide to the interpretation of the in situ results is provided.     

Gene expression analysis in sections and tissue microarrays of archival tissues by mRNA in situ hybridization

Altered expression of genes in diseased tissues can prognosticate a distinct natural progression of the disease as well as predict sensitivity or resistance to particular therapies. Archival tissues from patients with a known medical history and treatments are an invaluable resource to validate the utility of candidate genes for prognosis and prediction of therapy outcomes. However, stored tissues with associated long-term follow-up information typically are formalin-fixed, paraffin-embedded specimen and this can severely restrict the methods applicable for gene expression analysis. We report here on the utility of tissue microarrays (TMAs) that use valuable tissues sparingly and provide a platform for simultaneous analysis of gene expression in several hundred samples. In particular, we describe a stable method applicable to mRNA expression screening in such archival tissues. TMAs are constructed from sections of small drill cores, taken from tissue blocks of archival tissues and multiple samples can thus be arranged on a single microscope slide. We used mRNA in situ hybridization (ISH) on >500 full sections and >100 TMAs for >10 different cDNAs that yielded >10,000 data points. We provide detailed experimental protocols that can be implemented without major hurdles in a molecular pathology laboratory and discuss quantitative analysis and the advantages and limitations of ISH. We conclude that gene expression analysis in archival tissues by ISH is reliable and particularly useful when no protein detection methods are available for a candidate gene.     

Fluorescence in situ hybridization in paraffin tissue sections: pretreatment protocol

Fluorescence in situ hybridization has found wide application in the enumeration of gene and chromosome copy number both in isolated cells and in tissue sections. However, the technique has been less widely used than would be expected in formalin-fixed paraffin processed (archival) tissue. This article describes a method for assessing archival tissue sections, following pretreatment, before applying DNA probes, that gives consistent, reliable results.     

In situ hybridization technique to localize rRNA and mRNA in mammalian neurons

In situ hybridization provides a method for identifying cells that contain specific nucleic acid sequences. This report outlines an in situ hybridization procedure for mammalian neural tissue. The method maintains morphological quality and produces excellent specificity. Seven tritiated nucleic acid probes were examined: two ribosomal RNA probes, a control pBR322 plasmid probe, two probes encoding portions of the gene for oxytocin, one probe each encoding a portion of vasopressin glycoprotein, and neurophysin. Using cryostat-cut rat brain sections, rRNA probes labeled the cytoplasm of all cells and the nucleoli of larger neurons. The plasmid probe failed to produce a strong signal. Oxytocin and vasopressin probes appropriately labeled the cytoplasm of hypothalamic magnocellular neurons. Vasopressin parvocellular neurons were not identified by the current method, and the shorter length neurophysin probe failed to produce a signal. Methodological variables were examined by counting autoradiographic grains in cells. The longer oxytocin probe produced a stronger signal than the shorter oxytocin and vasopressin probes, and higher probe concentrations resulted in stronger signal. Hybridization could be abolished by tissue pretreatment with RNAse A, and longer exposure time increased signal strength. The outlined fixation steps with fresh-frozen tissue produced a superior signal compared to paraformaldehyde-perfused tissue.     

In situ RNA hybridization using Technovit resin in Arabidopsis thaliana

A protocol is described for RNA in situ hybridization using thin sections prepared by Technovit resin. Technovit is a widely used resin for histological examinations. Since it does not require time-consuming processes such as removal of the resin and can be performed without high temperature treatment, a high resolution of sections could be possible compared to other resins and paraffin. Thin sections (approximately 4 m) were made from inflorescences of Arabidopsis thaliana embedded in Technovit 8100 resin, and in situ hybridization was performed using the protocol described in this article. Hybridization signals were observed using LEAFY and other genes as probes, showing that this resin can be used for in situ analysis. In our experiments, the most important factor for a successful in situ hybridization pattern was to optimize the RNase A concentration after hybridization. We routinely used RNase A at a concentration of 2–5 ng/ml, a concentration much lower than that used for paraffin embedding method. Thus, the use of the Technovit resin for plant tissue embedding results in a faster protocol and greater quality than allowed by paraffin sections.     

In situ hybridization to mRNA: from black art to guiding light.

In situ hybridization to mRNA in embryo sections or wholemount embryos is one of the most powerful analytical tools available to the molecular developmental biologist. For many workers, this procedure provides the first insights into the function of newly isolated genes, allowing the formulation of hypotheses and setting the course for further research. This paper presents a personal historical perspective of the development of in situ hybridization, looks at the theory and practice of the technique, summarizes the current state of the art, and speculates on possible directions for the future as a tool in functional genomics.     

Sensitive nonradioactive detection of mRNA in tissue sections: novel application of the whole-mount in situ hybridization protocol.

The relative insensitivity of nonradioactive mRNA detection in tissue sections compared to the sensitive nonradioactive detection of single-copy DNA sequences in chromosome spreads, or of mRNA sequences in whole-mount samples, has remained a puzzling issue. Because of the biological significance of sensitive in situ mRNA detection in conjunction with high spatial resolution, we developed a nonradioactive in situ hybridization (ISH) protocol for detection of mRNA sequences in sections. The procedure is essentially based on the whole-mount ISH procedure and is at least equally sensitive. Increase of the hybridization temperature to 70C while maintaining stringency of hybridization by adaptation of the salt concentration significantly improved the sensitivity and made the procedure more sensitive than the conventional radioactive procedure. Thicker sections, which were no improvement using conventional radioactive ISH protocols, further enhanced signal. Higher hybridization temperatures apparently permit better tissue penetration of the probe. Application of this highly reliable protocol permitted the identification and localization of the cells in the developing heart that express low-abundance mRNAs of different members of the Iroquois homeobox gene family that are supposedly involved in cardiac patterning. The radioactive ISH procedure scarcely permitted detection of these sequences, underscoring the value of this novel method.     

High-throughput analysis of gene expression on tissue sections by in situ hybridization

Genome-scale sequencing projects, high-throughput RNAi screens, systematic gene targeting, and system-biology-based network predictions all depend on a validation of biological significance in order to understand the relevance of a particular finding. Such validation, for the most part, rests on low-throughput technologies. This article provides protocols that, in combination with suitable instrumentation, make possible a semi-automated analysis of gene expression on tissue sections by means of in situ hybridization. Knowledge of gene expression localization has the potential to aid, and thereby accelerate, the validation of gene functions.     

Non-isotopic in situ hybridization to detect chick Sox gene mRNA in plastic-embedded tissue

Summary In situ hybridization techniques have rapidly become widely used by the molecular biologist for the localization of specific nucleic acid sequences in individual cells or tissues. We describe the demonstration of Sox gene mRNA in chick tissue that has been embedded in the plastic methyl methacrylate to permit the preparation of sections for high-resolution light microscopy. Polymerization of the plastic was induced by using either N, N-dimethylaniline or N, N-3,5-tetramethylaniline. The in situ hybridization technique used was non-isotopic and used a digoxigenin-labelled probe detected with an antibody bound to alkaline phosphatase, which was then localized using X-phosphate-Nitro BT as a substrate-chromogen mix. Various pretreatments of the tissue sections were investigated, including the use of proteinase K, and heat-mediated techniques using a microwave oven and a pressure cooker. The best results were produced using pressure cooking on tissue in which the plastic had been chemically polymerized with N, N-3,5-tetramethylaniline. For the demonstration of Sox 11, this combination had a critical influence on the staining results, but for Sox 21 all protocols used produced good staining. This revised version was published online in November 2006 with corrections to the Cover Date.     

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

Summary We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding preserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electronmicroscopical level.     

In situ hybridization of semithin Epon sections with BrdU labelled oligonucleotide probes

We recently described a nonradioactive method for in situ hybridization with 5-bromo-2-deoxyuridine (BrdU) labelled oligonucleotide probes. An antibody to BrdU and immunocytochemistry were used in order to detect the hybridization signal. We have now applied this method to semithin Epon sections, in order to hybridize consecutive sections through single cells with different probes and to stain them with antibodies to neuropeptides. It could be shown that Epon embedding reserves mRNA well. In the present study we used a BrdU labelled synthetic oligonucleotide probe complementary to a fragment of the vasopressin precursor and an antibody to Arg-vasopressin. Vasopressin mRNA was demonstrable in a fraction of the vasopressin immunoreactive neurons in the magnocellular nuclei. In addition some of the magnocellular neurons showed either hybridization or vasopressin immunostaining only, perhaps indicating different stages of synthetic and secretory activity. The method described seems to be a valuable tool for studying synthetic activity in peptidergic neurons on a single cell level. The method might also have potential for in situ hybridization on the electron-microscopical level.