A methacrylate embedding procedure developed for immunolocalization on plant tissues is also compatible with in situ hybridization.

A recently described method that uses methacrylate embedding of aldehyde fixed plant tissues allows the immunolabelling of a range of antigens (Baskin et al. 1992). We have tested whether the same embedding procedure is also compatible with in situ hybridization. For this purpose we have used 2- 5 μm sections of methacrylate embedded plantlets of Arabidopsis thaliana. After removal of the resin the sections were prepared for in situ hybridization following standard procedures. Three different digoxygenin (dig)-labelled probes were used, recognizing RNAs coding for the chlorophyll a/b binding protein cab-140, the β-tubulin tub5 and meri a member of the meri-5 family. Each of the probes shows the labelling pattern expected from the literature. Moreover, the method allows a good structural preservation of very fragile tissues, in contrast to paraffin embedding. We conclude that methacrylate embedding, allowing both immunolabelling and in situ hybridization with high resolution and structural preservation, offers a high potential for the functional analysis of genes and proteins in plant development. This is especially true for Arabidopsis thaliana, a widely used model species where it seems to be the method of choice.     

In situ hybridization: general infectivity assay for retroviruses.

We have devised a general infectivity assay for retroviruses. A virus-specific [32P]DNA probe is hybridized in situ to a monolayer culture, and foci of infected cells in the monolayer are detected by exposure of the hybridized culture to X-ray films. The method is quantitative, in that it gives the same titer for Moloney murine leukemia virus as does the standard UV-XC test. The specificity of the assay is indicated by the fact that murine leukemia virus and baboon endogenous virus do not cross hybridize under the conditions used. The assay is completed within 1 to 3 weeks and should be broadly applicable for retroviruses which replicate without altering cellular morphology: its use is demonstrated with mouse mammary tumor virus and the helper virus of the reticuloendotheliosis complex.     

Localization of type 7 17beta-hydroxysteroid dehydrogenase in mouse tissues. In situ hybridization studies

The enzyme type 7 17beta-hydroxysteroid dehydrogenase (17beta-HSD) selectively catalyzes the conversion of estrone (E1) into estradiol (E2). In order to obtain detailed information about the exact sites of action of type 7 17beta-HSD, we have studied the cellular localization of type 7 17beta-HSD mRNA in mouse tissues using in situ hybridization (ISH). In parallel studies, we also measured the enzyme mRNA levels by quantitative real time (RT)-PCR. In the ovary, strong hybridization signal was restricted to corpus luteum cells. In the female mammary gland, type 7 17beta-HSD mRNA was found to be expressed in stromal cells surrounding the ducts. In the clitoral and preputial glands, specific labeling was observed in the epithelial cells of both acini and small ducts. In the adrenal gland, hybridization signal was observed in the zona fasciculata and reticularis in the cortex. In the liver, hybridization signal was found in all the hepatocytes. In the colon, type 7 17beta-HSD mRNA expression was restricted to epithelial cells of the mucosa. From the results obtained with quantitative real time RT-PCR, it appears, with a very few exceptions, that in tissues exhibiting low mRNA expression no ISH signal could be detected. The present data suggest that E2 can be formed through the action of type 7 17beta-HSD in specific cell types in the ovary and peripheral tissues, in addition to type 1 17beta-HSD, thus providing tissues with an alternative route of formation of E2.     

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.     

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.     

In situ hybridization study of the rice phloem thioredoxin h mRNA accumulation – possible involvement in the differentiation of vascular tissues

Thioredoxin (TRX) h was identified as a major protein in rice ( Oryza sativa L. cv. Kantou) phloem sap. Accumulation of TRX h mRNA in fully expanded rice leaves was detected only in the companion cells. To clarify the role of TRX h in phloem development, we performed in situ hybridization of TRX h mRNA in rice plants during the course of vascular development. TRX h mRNA was detected in all cell types at an early stage of development and became gradually confined to the companion cells in later stages. The possible roles of TRX h in vascular development are discussed.     

In situ hybridization and immunohistochemical analysis of cytochrome P450 1B1 expression in human normal tissues.

Cytochrome P450 1B1 (CYP1B1) is a recently cloned dioxin-inducible form of the cytochrome P450 supergene family of xenobiotic-metabolizing enzymes. CYP1B1 is constitutively expressed mainly in extrahepatic tissues and is inducible by aryl hydrocarbon receptor ligands. Human CYP1B1 is involved in activation of chemically diverse human procarcinogens, including polycyclic aromatic hydrocarbons and some aromatic amines, as well as the endogenous hormone 17 beta-estradiol. The metabolism of 17 beta-estradiol by CYP1B1 forms 4-hydroxyestradiol, a product believed to be important in estrogen-induced carcinogenesis. Although the distribution of CYP1B1 mRNA and protein in a number of human normal tissues has been well documented, neither the cells expressing CYP1B1 in individual tissue nor the intracellular localization of the enzyme has been thoroughly characterized. In this study, using nonradioactive in situ hybridization and immunohistochemistry, we examined the cellular localization of CYP1B1 mRNA and protein in a range of human normal tissues. CYP1B1 mRNA and protein were expressed in most samples of parenchymal and stromal tissue from brain, kidney, prostate, breast, cervix, uterus, ovary, and lymph nodes. In most tissues, CYP1B1 immunostaining was nuclear. However, in tubule cells of kidney and secretory cells of mammary gland, immunoreactivity for CYP1B1 protein was found in both nucleus and cytoplasm. This study demonstrates for the first time the nuclear localization of CYP1B1 protein. Moreover, the constitutive expression and wide distribution of CYP1B1 mRNA and protein in many human normal tissues suggest functional roles for CYP1B1 in the bioactivation of xenobiotic procarcinogens and endogenous substrates such as estrogens. (J Histochem Cytochem 49:229-236, 2001)     

In situ hybridization and immunolocalization of concentrative and equilibrative nucleoside transporters in the human intestine, liver, kidneys, and placenta

To better understand the role of human equilibrative (hENTs) and concentrative (hCNTs) nucleoside transporters in physiology and pharmacology, we investigated the regional, cellular, and spatial distribution of two hCNTs (hCNT1 and hCNT2) and two hENTs (hENT1 and hENT2) in four human tissues. Using in situ hybridization and immunohistochemical techniques, we found that the duodenum expressed hCNT1 and hCNT2 mRNAs in enterocytes and hENT1 and hENT2 mRNAs in crypt cells. In these cells, the hCNT and hENT proteins were predominantly localized in the apical and lateral membrane, respectively. Hepatocytes expressed higher levels of mRNAs of hENT1, hCNT1, and hENT2 than of hCNT2 and expressed all these proteins at hepatocyte cell borders and in the cytoplasm. While the kidney expressed hCNT1 and hCNT2 mRNAs in the proximal tubules, hENT1 and hENT2 mRNAs were present in the distal tubules, glomeruli, endothelial cells, and vascular smooth muscle cells. Proximal tubules adjacent to corticomedullary junctions expressed hENT1, hCNT1, and hCNT2 mRNA. Immunolocalization studies revealed predominant localization of hCNTs in the brush-border membrane of the proximal tubular epithelial cells and hENTs in the basolateral membrane of the distal tubular epithelial cells. Chorionic villi sections of human term placenta expressed mRNAs and proteins for hENT1 and hENT2 but only mRNA for hCNT2. Immunolocalization studies showed presence of hENT1 in the brush-border membrane of the syncytiotrophoblasts. These data are critical for a better understanding of the role of nucleoside transporters in the physiological and pharmacological effects of nucleosides and nucleoside drugs, respectively.     

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.     

In situ hybridization using PEG-embedded tissue and riboprobes: increased cellular detail coupled with high sensitivity

We describe a procedure for preparing tissue sections by embedding in polyethylene glycol for subsequent in situ hybridization analysis using single-stranded RNA probes. Improved tissue morphology is obtained as compared to frozen sections, and the embedding procedure is milder and faster than paraffin embedding. Sections as thin as 2 microns are readily cut from PEG-embedded brain tissue. A simplified hybridization protocol (Clayton et al.: Neuron 1:249, 1988) supports the detection of even low-abundance brain mRNAs (less than or equal to 10(-4) fractional mRNA mass). By employing high stringency washes in place of ribonuclease treatment after hybridization, cell RNA is retained for cresyl violet staining, and high signal:noise ratios are achieved. Solutions to problems with section mounting and adherence to glass slides are presented. The combination of improved morphology, high signal levels, and relative simplicity should make this procedure useful in a variety of applications.     

Decay of Branchiostoma: implications for soft-tissue preservation in conodonts and other primitive chordates

Decay experiments on the cephalochordate Branchiostoma lanceolatum ('amphioxus') demonstrate that the most decay resistant structures are the notochord sheath and the cartilaginous rods which support the gill bars. However, even more labile soft parts, such as the muscles and skin may survive for at least 124 days under totally anoxic conditions. As the chevron-shaped muscles of the myomeres shrink and collapse, those on opposite sides of the trunk maybe displaced, resulting in pronounced offsetting. Only 1.42% of the initial dry weight of Branchiostoma is resistant to alkali and acid hydrolysis, compared to 46% in the polychaete Nereis virens. Branchiostoma is only likely to be fossilized as a result of decay inhibition and replication by early diagenetic minerals. The results of these experiments cast light on the interpretation of a number of primitive fossil chordates. There is no reason to infer extracellular decay-resistant cuticle in the Burgess Shale Pikaia. The axial lies preserved in the conodont animal specimens from the Carboniferous of Edinburgh, Scotland, represent the notochord. The displacement of the elements to one side of the head reflects the true position of the apparatus - the surrounding tissue has been lost through decay. The chevron-shaped structures in the Carboniferous chordate Conopiscius are the muscles of the myomeres, not external scales. The lines delineating the segments in the Silurian Jamoytius most likely represent the myosepta. There is some doubt about the nature of the only specimen interpreted as a fossil cephalochordate, Palaeobranchiostoma hamatotergum from the Permian of South Africa. □ Taphonomy, decay, softparts, Cephalochordata , Branchiostoma, lancelet, Chordata , Pikaia, conodont , Conopiscius, Jamoytius, Palaeobranchiostoma.     

Fluorescent in situ hybridization on tissue microarrays: challenges and solutions

Tissue microarray (TMA) technology has provided a high throughput means of evaluating potential biomarkers and therapeutic targets in archival pathological specimens. TMAs facilitate the rapid assessment of molecular alterations in hundreds of different tumours on a single slide. Sections from TMAs can be used for any in situ tissue analysis, including fluorescent in situ hybridization (FISH). FISH is a molecular technique that detects numerical and structural abnormalities in both metaphase chromosomes and interphase nuclei. FISH is commonly used as a prognostic and diagnostic tool for the detection of translocations and for the assessment of gene deletion and amplification in tumours. Performing FISH on TMAs enables researchers to determine the clinical significance of specific genetic alterations in hundreds of highly characterized tumours. The use of FISH on archival paraffin embedded tissues is technically demanding and becomes even more challenging when applied to paraffin embedded TMAs. The problems encountered with FISH on TMAs, including probe preparation, hybridization, and potential applications of FISH, will be addressed in this review.     

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.     

Fluorescence in situ hybridization for phytoplasma and endophytic bacteria localization in plant tissues

In the present study, we developed a rapid and efficient fluorescence in situ hybridization assay (FISH) in non-embedded tissues of the model plant Catharanthus roseus for co-localizing phytoplasmas and endophytic bacteria, opening new perspectives for studying the interaction between these microorganisms.     

Decaying in different clays: implications for soft‐tissue preservation

Lagerstätten, places where soft‐bodied organisms became mineralized, provide a substantial bulk of palaeobiological information, but the detailed mechanisms of how soft‐tissue preservation takes place remain debatable. An experimental taphonomy approach, which allows for direct study of decay and mineralization, offers a means to study the preservational potential of different soft‐bodied organisms under controlled conditions. Here we compare the preservational capacity of two types of clay (kaolinite and montmorillonite) through a long‐term (24 month) experiment involving the burial and decay of small crustaceans. Our experimental design is innovative in that it models catastrophic sedimentation in fine‐grained colloidal suspension, which is believed to form Lagerstätten deposits. We demonstrated better preservation of buried organisms in clays compared to water, and in kaolinite compared to montmorillonite. As aluminium cations were present in high concentrations in kaolinite sediment but not in montmorillonite, the better preservation in kaolinite is attributed to the tanning properties of aluminium, which catalyses cross‐linking in proteins, protecting them from bacterial degradation. Anaerobic environments and acidification also slow down decay, but they are less effective than tanning. Kaolinite and montmorillonite replaced the crustacean integuments differently: in the remains buried in kaolinite, Al and Si were detected in equal proportions, while in those buried in montmorillonite, the Si content appeared to be much higher even in comparison with the initial sample of the clay. These variations probably arose from the different dynamics of acidic hydrolysis in the two clays associated with anaerobic decomposition of organic matter. Our results show that the preservation mechanism includes multi‐component interactions between the solution, mineral, sediment and organic remains; taken separately, any single component explains little. The specific conditions that occur within the colloidal clay sediments can facilitate conservation and start fast mineralization according to chemical properties and elemental content.