Double in situ hybridization for detection of Herpes simplex virus and cytomegalovirus DNA using non-radioactive probes.

We describe a double in situ hybridization assay for the simultaneous detection of Herpes simplex virus (HSV) and cytomegalovirus (CMV) DNA in infected cell cultures using non-radioactive-labeled probes. This work used a biotinylated HSV DNA probe, which can be revealed by an avidin-biotin-peroxidase complex and a digoxigenin-labeled CMV DNA probe, visualized by anti-digoxigenin F(ab) fragments conjugated with alkaline phosphatase. Light microscopy visualization was achieved by the contrasting colors of appropriate peroxidase and alkaline phosphatase reaction products (red and dark blue, respectively). The time required to perform the double hybridization assay was about 3 hr. This double hybridization assay proved to be sensitive, specific, and provided good resolving power.     

Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes

Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF- or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidin-biotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV16 DNA and haptenized HSV2 DNA.     

Two colour DNA in situ hybridization for the detection of two viral genomes using non-radioactive probes

Summary Methods for the simultaneous detection of two virus types in cytological preparations or tissue sections by non-radioactive in situ hybridization were investigated. As a model system, CaSki cells, which have human papilloma virus type 16 (HPV 16) DNA integrated in their cellular genome, were in vitro infected with Herpes simplex virus 2 (HSV2). DNA probes for both viruses were labeled with biotin, acetylaminofluorene (AAF), and transaminated-sulfonated cytosine (TS-modified). Best results were obtained when a mixture of biotinated and haptenized DNA probes (AAF-or TS-modified) was used for hybridization. The biotinated hybrid was demonstrated with a streptavidinbiotinated alkaline phosphatase staining reaction, whereas the haptenized hybrid was visualized by an indirect peroxidase method. Visualisation of both viral DNAs in the same cell was possible by a combination of biotinated HPV 16 DNA and haptenized HSV2 DNA.     

Simple chemical synthesis and hybridization properties of non-radioactive DNA probes

A simple chemical method for the synthesis of non-radioactive DNA probes is described: triazolyl-containing sequences were built by incorporation of 4-triazolylpyrimidin-2-ones instead of cytidines during oligodeoxyribonucleotide synthesis. The activating triazolyl groups were then displaced by a diamine which was further derivatized by a label, such as biotin. Synthesized DNA probes were oligonucleotides complementary to a cloned human antithrombin III DNA sequence. These probes, containing the same label at different positions of the sequence, were hybridized to their target DNA immobilized on nitrocellulose. Their hybridization specificity and stability were studied. Hybrid detection was performed either colorimetrically by the streptavidin-alkaline phosphatase-based system or by autoradiography after 5'-32P labeling of the probes: 15 fmol (0.05 microgram) of complementary sequence could be visualized in the two cases.     

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     

Mercurated nucleic acid probes, a new principle for non-radioactive in situ hybridization

This report describes the localization of specific nucleic acid sequences in interphase nuclei and metaphase chromosomes by a new hybridocytochemical method based on the use of mercurated nucleic acid probes. After the hybridization a sulfhydryl-hapten compound is reacted with the hybrids formed. A number of such ligands were synthesized and tested. A fluorescyl ligand could be used for the direct visualization of highly repetitive sequences. For indirect immunocytochemical visualization trinitrophenyl ligands were found to be more sensitive than biotinyl analogues. These ligands were applied for the detection of target sequences in metaphase chromosomes and interphase nuclei of somatic cell hybrids, human lymphoid cell lines and blood cell cultures. The sequences were in the range of high to low copy numbers. The lower limit of sensitivity is indicated by the visualization of two human unique DNA fragments (40 and 15.6 kb) in human metaphases. The method is rapid, gives consistent results and can be used for both RNA and DNA probes. Other potentials of the new principle are discussed.     

A non-radioactive in situ hybridization method based on mercurated nucleic acid probes and sulfhydryl-hapten ligands.

Mercurated nucleic acid probes can be used for non-radioactive in situ hybridization. The principle of the method is based on the reaction of the mercurated pyrimidine residues of the in situ hybridized probe with the sulfhydryl group of a ligand which contains a hapten. Next, the hapten is immunocytochemically detected. Previous experiments showed that stable coupling of the sulfhydryl ligands could only be obtained when positively charged amino groups are present in the ligand. On basis of this finding, ligands were synthesized containing a sulfhydryl group, two lysyl residues and hapten groups such as trinitrophenyl, fluorescyl and biotinyl. The ligands, free or bound to mercurated nucleic acids, were immunochemically characterized in ELISAs. The method was shown to be specific and sensitive in the detection of target DNA in situ on microscopic preparations and in dot-blot hybridization reactions on nitrocellulose.     

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)     

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.     

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.     

In situ hybridization with non-radioactive digoxigenin-11-UTP-labeled cRNA probes: localization of developmentally regulated mouse tenascin mRNAs.

An improved method for in situ hybridization was developed in order to identify the tissue-specific expression of messenger RNA (mRNA) for the novel extracellular matrix glycoprotein, tenascin, during mouse development. Non-radioactive RNA probes were generated by incorporating digoxigenin-11-UTP instead of conventional isotopic labels. Hybridization of anti-sense probes to complementary mRNAs was detected by a chromogenic staining reaction catalyzed by an anti-digoxigenin antibody-alkaline phosphatase conjugate. Markedly improved enhancement of staining was achieved by expanding the complexity of probes and strictly controlling the degree of proteolytic digestion of paraformaldehyde-fixed tissue sections. Six different complementary RNA (cRNA) probes representing most of the tenascin mRNA sequence were prepared. Very weak signals were obtained after single applications of each probe, but strong specific signals were present when all six probes were mixed together. In either case, no signal was found without prior proteolytic digestion of tissue sections with proteinase K. Treatment with increasing concentrations of proteinase K initially resulted in increased sensitivity of signal detection, but extensive digestion resulted in histological sections of poor quality for light microscopy. Optimal conditions varied according to the tissue type examined. In lung, in situ hybridization detected tenascin mRNA in the relatively large cells lining alveolar walls adjacent to type I pneumocytes. In cerebellum, glial cells of the Purkinje cell layer contained tenascin mRNA, but Purkinje cells did not. In both cases, hybridization signals were confined to the cytoplasm of cells, and no extracellular staining was observed. This method provides a promising new tool for analysis of spatio-temporal regulation of tenascin gene expression during embryogenesis and oncogenesis.     

Effect of unlabeled helper probes on detection of an RNA target by bead-based sandwich hybridization

Unlabeled helper oligonucleotides assisting a bead-based sandwich hybridization assay were tested for the optimal placement of the capture and detection probes. The target used was a full-length in vitro synthesized mRNA molecule. Helper probes complementary to regions adjacent to the binding site of the 5' end attached capture probe were found much more effective than helper probes targeting positions adjacent to the detection probe binding site. The difference is believed to be caused by a disruption of the RNA secondary structure in the area where the capture probe binds, thereby reducing structural interference from the bead. The use of additional helpers showed an additive effect. Using helpers at both sides of the capture and detection probes showed a 15- to 40-fold increase in hybridization efficiency depending on the target, thereby increasing the sensitivity of the hybridization assays. Using an electrical chip linked to the detection probe for the detection of p-aminophenol, which is produced by alkaline phosphatase, a detection limit of 2 x 10(-13) M mRNA molecules was reached without the use of a nucleic acid amplification step.     

Cellular localization of induced human interferon-beta mRNA by non-radioactive in situ hybridization

Induced interferon-beta (IFN-beta) mRNA was localized in human FS-4 fibroblasts by in situ hybridization using biotinylated probes. The hybridization sites were detected by incubation with a nick-translated genomic DNA probe (1.8 kb) via streptavidin-colloidal gold followed by silver contrast enhancement. The positive signals were observed by reflection-contrast light microscopy. IFN-beta mRNA was transiently induced by poly r(I): r(C) in fibroblasts 2-4 h after induction. Induction in the presence of cycloheximide and actinomycin D (superinduction conditions) exhibited an enhanced level of IFN-beta mRNA with a maximum at 4-8 h. The kinetics of the IFN-beta mRNA expression in the cytoplasm as revealed by in situ hybridization proved to be compatible with the results of Northern blotting experiments of total cellular RNA.     

Adaptation of a non-radioactive in situ hybridization method to electron microscopy: detection of tenascin mRNAs in mouse cerebellum with digoxigenin-labelled probes and gold-labelled antibodies

In this study we describe a method for the detection of mRNAs at the ultrastructural level using a non-radioactive in situ hybridization method based on digoxigenin-labelled cRNA probes and gold-labelled digoxigenin-specific antibodies. We applied this protocol to an analysis of the expression of the extracellular matrix protein tenascin in the developing cerebellar cortex of the mouse. To gain an impression of the sensitivity attainable with digoxigenin-labelled probes, we first established at the light microscopic level that the hybridization signal obtained with the non-radioactive probe is as sensitive as that obtained with a ³⁵S-labelled probe. The non-radioactive hybridization protocol was then combined with electron microscopic post-embedding and immunogold detection techniques. Tenascinspecific, digoxigenin-labelled cRNA probes were hybridized to ultrathin sections of Lowicryl K4M-embedded tissue and the probe/target mRNA hybrids were detected using gold-labelled antibodies to digoxigenin. In agreement with the observations from in situ hybridization at the light microscopic level, specific labelling was observed in Golgi epithelial cells in the region of the Purkinje cell layer and cells in the internal granular layer, which could be identified as astrocytes by ultrastructural criteria. Labelling was detectable in association with free ribosomes and ribosomes of the rough endoplasmic reticulum. In addition, focal hybridization signals were occasionally found in the nucleus. No signal was observed in Golgi epithelial cells or astrocytes using sense or in any other cerebellar cell type using either sense or anti-sense probes. The described in situ hybridization technique uses ultrastructural criteria to associate the presence of a given mRNA species with a particular cell type. Additionally, it provides information about the target mRNA's subcellular distribution, thus offering the possibility to study intracellular transport of particular mRNAs.     

High-density hapten labeling and HRP conjugation of oligonucleotides for use as in situ hybridization probes to detect mRNA targets in cells and tissues.

Oligonucleotides that carry a detectable label can be used to probe for mRNA targets in in situ hybridization experiments. Oligonucleotide probes (OPs) have several advantages over cDNA probes and riboprobes. These include the easy synthesis of large quantities of probe, superior penetration of probe into cells and tissues, and the ability to design gene- or allele-specific probes. One significant disadvantage of OPs is poor sensitivity, in part due to the constraints of adding and subsequently detecting multiple labels per oligonucleotide. In this study, we compared OPs labeled with multiple detectable haptens (such as biotin, digoxigenin, or fluorescein) to those directly conjugated with horseradish peroxidase (HRP). We used branching phosphoramidites to add from two to 64 haptens per OP and show that in cells, 16-32 haptens per OP give the best detection sensitivity for mRNA targets. OPs were also made by directly conjugating the same oligonucleotide sequences to HRP. In general, the HRP-conjugated OPs were more sensitive than the multihapten versions of the same sequence. Both probe designs work well both on cells and on formaldehyde-fixed, paraffin-embedded tissues. We also show that a cocktail of OPs further increases sensitivity and that OPs can be designed to detect specific members of a gene family. This work demonstrates that multihapten-labeled and HRP-conjugated OPs are sensitive and specific and can make superior in situ hybridization probes for both research and diagnostic applications.     

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.     

Preparation of nonradioactive probes for in situ hybridization

In situ hybridization (ISH) enables the precise localization of RNA targets and provides an avenue to study the temporal and spatial patterns of expression of specific genes. ISH has evolved from being an esoteric technique to one that is routinely used by researchers in many areas of research. A major driving force has been the development of numerous nonisotopic labeling and signal detection methods. Historically, radioactive probes and autoradiography provided sensitivity that was unattainable with nonisotopic probes. But the long exposure times required for signal detection and the perceived dangers associated with radioactivity limit its use. Advances in nonisotopic detection systems have overcome many of the limitations associated with using radiolabeled probes. One of the most significant contributions from nonisotopic methods is the ability to discriminate between multiple nucleic acid sequences simultaneously.