Detection of nerve growth factor and its mRNA by separate and combined immunohistochemistry andin situ hybridization in mouse salivary glands

Summary Intense labelling of secretory cells in the male mouse submandibular gland was observed afterin situ hybridization using mouse nerve growth factor (NGF) cDNA probes. Under the same conditions, sparse less intensely labelled cells were also found in the sublingual gland. Hybridization to a chicken NGF cDNA probe gave weak labelling on the glands in accordance with a weak cross-hybridization between mouse NGF mRNA and chicken NGF cDNA probes, whereas no labelling was seen using pUC9 DNA as a hybridization probe. A combination ofin situ hybridization and immunohistochemistry was also carried out on the same sections of submandibular gland. A good correlation was seen between actively synthesizing and intensely immunoreactive cells in the gland. The technique described here allows the detection of individual cells synthesizing relatively low levels of NGF. The combination ofin situ hybridization and immunocytochemistry on the same section should be particularly useful in cases where NGF is transported away from its site of synthesis.     

A nonradioactive whole-mount in situ hybridization protocol for Porphyra (Rhodophyta) gametophytic germlings

The objective of this study was to develop a method for whole-mount in situ hybridization (WISH) by using elongation factor-1 (EF-1) riboprobes in the red alga Porphyra yezoensis Ueda. Several modifications to the general WISH protocol, such as use of a short-length probe, performing partial digestion of the cell wall, optimization of proteinase K concentration and additional washing steps after hybridization were essential to reduce non-specific staining and to obtain sufficient quality of data. This protocol made it possible to detect a specific signal as a positive control in WISH assays of P. yezoensis.     

Procedure for whole mount fluorescence in situ hybridization of interphase nuclei on Arabidopsis thaliana

A procedure for whole mount fluorescence in situ hybridization (FISH) on plant tissue is reported. The technique was demonstrated on seedlings and flowers of Arabidopsis thaliana L. with rDNA as a probe, labelled, both for direct and indirect detection. It was found that fixation in 1% formaldehyde yielded the best results with respect to morphology and hybridization efficiency. The combination of whole mount FISH and confocal scanning laser microscopy allowed the nuclear localization of the rDNA loci in all tissues of both seedlings and flowers. Direct labelling yielded the best signal-to-noise ratio, especially in the apical zones of the seedlings. The technique was further illustrated on seedlings of A. thaliana in double labelling experiments with rDNA and a tandemly repeated, 500 bp sequence of A. thaliana. Although nuclei in all tissues in the seedling exhibited both signals, hybridization efficiency for both signals was reduced in the dense, apical zones as compared with single labelling experiments with rDNA.     

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.     

Digoxigenin as a probe label for in situ hybridization on skeletal tissues.

Summary Non-specific staining was encountered using digoxigenin-labelled cDNA probes forin situ hybridization on sections of skeletal tissues. This staining was most pronounced in cartilaginous matrices. Experimental procedures indicate that the background staining is caused by antibody-binding to hydrophobic sites in the tissues revealed by proteolytic permeabilization. A protocol for minimizing this background is described.     

The effects of varying key steps in the non-radioactive in situ hybridization protocol: a quantitative study

Summary In situ hybridization represents a major advance in the study of gene expression and, thus, in the evaluation of cellular function in histological sections. The availability of oligonucleotide probes labelled with biotin and sensitive immunohistochemical detection systems makes the study of different types of mRNA by in situ hybridization easier. However, a large number of protocols have been reported, which is sometimes confusing. The present study analyses quantitatively the influence of each important step of in situ hybridization on the staining intensity of rat proinsulin mRNA. The aim was to optimize technical conditions, to make the method sensitive and to evaluate its reproducibility for proinsulin mRNA detection and measurements. The duration of fixation and the digestion have an important impact on the results. The optimal digestion time depends on the fixation. With a digestion of 30 g ml^–1 proteinase K for 12 min at 37°C, the optimal fixation time was 24 h. Section thickness also influences the staining intensity. The intensity of the staining increases as the section thickness increases from 3 to 5 m before slowly decreasing. A weak paraformaldehyde post-fixation (0.4% for 20 min) gives best results in comparison to a stronger post-fixation (4% for 10 min). An increase of probe concentration leads to a higher specific labelling, reaching a plateau at 800 ng ml^–1. Hybridization temperature (37–42°C) exerts little influence. However, the temperature of the washes and the immunodetection system have a major effect on the intensity of labelling.Quantification has permitted the evaluation of the influence of each key for optimizing and standardizing the non-radioactive in situ hybridization protocol. In these well-defined conditions, the intra and inter-assay coefficient of variation remains lower than 6% and thus the method can be used to quantify the content of proinsulin mRNA or other specific mRNAs in experimental and pathological conditions.     

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.     

Bone sialoprotein mRNA expression and ultrastructural localization in fetal porcine calvarial bone: comparisons with osteopontin

Summary Bone sialoprotein (BSP) and osteopontin (OPN) are two major non-collagenous proteins in bone that have similar biochemical properties and can mediate cell attachment through an RGD (Arg-Gly-Asp) motif that recognizes the vitronectin receptor. To facilitate evaluations of the biological functions of BSP and OPN in bone formation, affinity-purified rabbit polyclonal antibodies against porcine BSP and OPN were used, together with a high-resolution protein A-gold immunocytochemical technique to reveal the ultrastructural localization of these proteins in undermineralized sections of 50-day fetal porcine calvarial bone. In addition,³⁵S-labelled antisense riboprobes were prepared to demonstrate the cellular expression of BSP and OPN in the same tissues usingin situ hybridization. Immunolocalization for both BSP and OPN revealed the highest density of gold particles associated with electron-dense organic material found at the mineralization front and in ‘cement lines’. Labelling was also observed in the mineralized matrix over electron-dense material between collagen fibrils. In the osteoid of newly-formed bone, immunogold labelling for BSP and OPN was associated with loci of mineralization, which were often characterized by feathery clusters of fine needle-like crystals. Results ofin situ hybridization on the same tissues demonstrated that BSP mRNA expression was restricted to differentiated osteoblasts with particularly strong signals evident at sites ofde novo bone formation. More moderate expression of BSP was observed in ‘older’ osteoblasts and in some of the newly-entrapped osteocytes. Although expression of OPN mRNA was also observed in osteoblasts and osteocytes, the level of hybridization was similar for most bone cells and not markedly stronger than the signal observed in some stromal cells. While it is evident from these and other studies that both BSP and OPN are associated with bone formation, the differences observed in cellular expression indicate distinct roles for these proteins in bone formation.     

Microwave irradiation in label-detection for diagnostic DNA-in situ hybridization.

Summary A DNA-in situ hybridization protocol was adapted for application to sections of routinely processed paraffin embedded material. This protocol was developed previously for detecting DNA-virus infected cells in whole cell preparations and employs biotinylated DNA as probe. Three different biotin detection methods were optimized and applied. The first uses streptavidin and a biotinylated complex of alkaline phosphatase, the second consists of an immunogold-silver staining, and the third of a peroxidase technique using a silver amplification. The alkaline phosphatase method was the most rapid, and as sensitive as the immunogold-silver staining. The peroxidase method was the most sensitive. Microwave irradiation was applied to the different incubation steps of these three detection methods. Short incubations with microwave irradiation gave very poor results when peroxidase labelled antibodies were used. Short incubation with microwave irradiation gave results comparable to those obtained with conventional incubations, when streptavidin, antibiotin, complexed alkaline phosphatase, or gold labelled goat antirabbit were used. It was thus shown that microwave irradiation creates the possibility of a very rapid label-detection for nonradioactive DNA-in situ hybridization.     

The use of microwave irradiation as a pretreatment toin situ hybridization for the detection of measles virus and chicken anaemia virus in formalin-fixed paraffin-embedded tissue

Summary Microwave irradiation was investigated as a pretreatment toin situ hybridization on formalin-fixed, paraffin-embedded tissue. Two probe/tissue systems were used: a single-stranded RNA probe for the detection of measles virus nucleocapsid genome in subacute sclerosing panencephalitis brain tissue, and a double stranded DNA probe for chicken anaemia virus in thymus of chicken infected with the virus. Microwaving, when used as sole pretreatment, was not as effective as the more traditional enzyme pretreatments forin situ hybridization. However, when used in combination with existing pretreatments, a significant increase was found in hybridization signal in both brain and thymus tissue. This was emphasized when combination enzyme/microwave pretreatments were used prior to detection of measles virus byin situ hybridization in a series of five archival subacute sclerosing panencephalitis cases. The use of microwave irradiation would be recommended as a means of supplementingin situ hybridization methods, especially when using long-term formalin fixed paraffin-embedded tissue.     

sRNA‐FISH: versatile fluorescent in situ detection of small RNAs in plants

Localization of mRNA and small RNAs (sRNAs) is important for understanding their function. Fluorescent in situ hybridization (FISH) has been used extensively in animal systems to study the localization and expression of sRNAs. However, current methods for fluorescent in situ detection of sRNA in plant tissues are less developed. Here we report a protocol (sRNA‐FISH) for efficient fluorescent detection of sRNAs in plants. This protocol is suitable for application in diverse plant species and tissue types. The use of locked nucleic acid probes and antibodies conjugated with different fluorophores allows the detection of two sRNAs in the same sample. Using this method, we have successfully detected the co‐localization of miR2275 and a 24‐nucleotide phased small interfering RNA in maize anther tapetal and archesporial cells. We describe how to overcome the common problem of the wide range of autofluorescence in embedded plant tissue using linear spectral unmixing on a laser scanning confocal microscope. For highly autofluorescent samples, we show that multi‐photon fluorescence excitation microscopy can be used to separate the target sRNA‐FISH signal from background autofluorescence. In contrast to colorimetric in situ hybridization, sRNA‐FISH signals can be imaged using super‐resolution microscopy to examine the subcellular localization of sRNAs. We detected maize miR2275 by super‐resolution structured illumination microscopy and direct stochastic optical reconstruction microscopy. In this study, we describe how we overcame the challenges of adapting FISH for imaging in plant tissue and provide a step‐by‐step sRNA‐FISH protocol for studying sRNAs at the cellular and even subcellular level.     

In situ hybridization technique using an immunogold silver staining system

Summary Anin situ hybridization technique using an immunogold silver staining detection system was used to detect biotinylated DNA probes in cultured cells and in formalin-fixed paraffin-embedded tissue. The detection method is rapid, reliable and economical, producing an insoluble signal at the site of hybridization which is visible at low-power light microscopy and which can be enhanced by epipolarization microscopy.     

Translocation events demonstrated by molecular,in situ hybridization and chromosome pairing analyses in highly asymmetric somatic hybrid plants

Cytological analyses show rearranged chromosomes in some highly asymmetric nuclear hybrids obtained after fusion of mesophyll protoplasts ofNicotiana plumbaginifolia (wild type) with γ-irradiated (100 krad), kanamycin-resistant mesophyll protoplasts ofPetunia hybrida. Molecular, cytogenetic andin situ hybridization analyses performed on the asymmetric somatic hybrid P₁, previously identified as having a clearly metacentric chromosome besides a nearly completeNicotiana chromosome complement, are reported. Meiotic analysis andin situ hybridization experiments using ribosomal DNA as a probe showed that this metacentric chromosome represents a translocation of a chromosome fragment onto chromosome 9 ofN. plumbaginifolia. Southern hybridization with an rDNA probe showed that onlyNicotiana-specific rDNA was present.In situ hybridization experiments, using total genomic DNA ofP. hybrida as a probe, demonstrated that the translocated fragment representedPetunia DNA.     

A new method to perform nonradioactive tissue printing using digoxigenin-labeled riboprobes

We describe a modified protocol to perform a tissue print northern using digoxigenin-labeled riboprobes. The result of the hybridization is directly visualized on the membrane by an alkaline phosphatase detection system.     

Histone H3 mRNA in situ hybridization for identifying proliferating cells in human pancreas, with special reference to the ductal system

In general, the incidence of proliferating cells parallels that of carcinogenesis. We have investigated proliferating activity and phenotype expression in epithelial cells in normal tissue, mucinous metaplasia and ductal adenocarcinoma of the pancreas. Twenty-eight resected pancreases (15 cases of pancreatic ductal adenocarcinoma and 13 cases of other diseases) were examined. Formalin-fixed, paraffin-embedded tissue sections were examined for proliferating cell activity using histone H3 mRNA in situ hybridization and immunostaining for Ki-67. In the normal pancreas, the labelling indices for proliferating cells were low and no generating zone was found. The following progressive increase was found in the labelling indices: normal ductal epithelium < mucinous metaplasia without papillary hyperplasia < mucinous metaplasia with papillary hyperplasia < ductal carcinoma. In the pancreatic ductal adenocarcinomas, the S-phase fraction, as defined by the ratio H3-mRNA-labelling index/Ki-67-labelling index, increased as the degree of differentiation decreased. Mucinous metaplasia with papillary hyperplasia showed organoid differentiation toward pyloric mucosa. If used in combination with other proliferative markers on paraffin-embedded tissue sections, histone H3 mRNA in situ hybridization could open broader perspectives on the biology of cell proliferation in the pancreatic ductal system.     

Simultaneous in situ hybridization and TUNEL to identify cells undergoing apoptosis

Apoptotic cells in tissue sections can be localized by in situ labelling of partly degraded DNA. In a heterogeneous population of cells, however, the specific identity of cell types undergoing apoptosis often cannot be reliably achieved at the light microscope level because of the marked alterations in cellular morphology that characterize apoptosis. In order to clearly specify cell types undergoing apoptosis, in situ end labelling has been coupled to immunohistochemistry. This method is limited by the availability of antibodies that bind to cell-specific protein markers in tissue sections. In contrast, we describe a method that combines in situ end labelling with in situ hybridization, a technique that specifies cell types based on mRNA expression. Taking advantage of the specific expression of surfactant protein C mRNA in type II alveolar epithelial cells, we demonstrate that this technique has the ability to localize alveolar type II cells undergoing apoptosis in vivo after the intratracheal instillation of an antibody that activates the cell surface Fas protein. The wide availability of cell-specific gene markers suggests that this method can be adapted to define cell types that undergo apoptosis during various physiological and pathological states in vivo. This revised version was published online in November 2006 with corrections to the Cover Date.     

Ultrastructural in situ hybridization: A review of technical aspects

Detection of nucleic acid sequence at the ultrastructural level has allowed us to better understand the expression of genes in some fields of application in cell biology. In situ hybridization at the ultrastructural level can be carried out using three different methods: on vibratome sections before embedding in epoxy resin, on ultrathin frozen section, or on ultrathin section of tissue embedded in hydrophilic resin such as Lowicryl. Before starting the detection of nucleic acid sequences at the electron microscope level, the experimenter has to choose various parameters: the type of tissue fixation, the probe and its label, and the in situ hybridization method, depending on the sensitivity, the resolution and the ultrastructural preservation required. This review of technical aspects, by describing the different methods of ultrastructural in situ hybridization, will help the experimenter to optimize each step of the hybridization procedure.     

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.