Search

Whole-mount in situ hybridization (WISH) is a highly sensitive method for spatial and temporal visualization of RNA transcripts throughout an entire tissue. This method is an excellent tool for studying both embryonic development and disease. It is based on sequence complementation between a Digoxigenin (DIG)-labeled antisense RNA probe and the sense RNA transcript of interest in the tissue. The labeled probe is then visualized by immunohistochemical methods using an antibody conjugated to alkaline phosphatase that catalyzes a color reaction.

In situ hybridization (ISH) is a type of hybridization that uses a labeled complementary DNA or RNA strand (i.e., probe) to localize a specific DNA or RNA sequence in a portion or section of tissue (In Situ) or in the entire tissue (whole mount ISH). Localization of endogenous transcripts is a desirable approach for confirming expression patterns. This is distinct from immunohistochemistry, which usually localizes proteins in tissue sections. DNA ISH can be used to determine the structure of chromosomes. However, RNA ISH (hybridization histochemistry) is used to measure and localize mRNAs and other transcripts within tissue sections or whole mounts. RNA-RNA hybrids approach may offer increased sensitivity, which is more stable than that of DNA-RNA hybrids. Here we describe the efficient ISH protocol for nonradioactive (i.e., in direct methods using digoxigenin (DIG) system) RNA probes, and it can be performed in less than 3 days.

We developed a simple, direct, and cost-effective approach to search for the most likely target genes of a known miRNA in vitro. We term this method "Labeled microRNA (miRNA) pull-down assay system," or LAMP. Briefly, the pre-miRNA is labeled with digoxigenin (DIG), mixed with cell extracts, and immunoprecipitated by anti-DIG antiserum. We concluded that LAMP is an experimental approach for high-throughput identification of the target gene of known miRNAs from both Caenorhabditis elegans and zebrafish (Danio rerio), yielding fewer false-positive results than those produced by using only the bioinformatics approach.

The protocol reported in this chapter describes a method for the detection and spatial localisation of microRNAs (miRNAs) in cryopreserved primary leukaemic suspension cells using digoxigenin (DIG)-labelled, Locked Nucleic Acid (LNA)-modified probes, and fluorescence in situ hybridisation (FISH). The LNA probe hybridisation yields highly accurate signals able to discriminate between single nucleotide differences and hence between closely related miRNA family members. DIG-labelled LNA probes for mature miRNAs are detected using an anti-DIG fluorescein isothiocyanate (FITC) conjugated antibody and the fluorescent signals visualised with a confocal microscope, which permits the spatial localisation of the miRNAs. Using LNA-FISH, we visualised the spatial localisation of two mature miRNAs, miR-127 and miR-154, in primary acute myeloid leukaemia (AML) suspension cells, and thus, we confirmed their expression in a specific leukaemic subtype as measured by real-time PCR.

Methods are described for the cellular localization of expression of flavin-containing monooxygenase (FMO) genes in various mouse tissues by in situ hybridization. These include the production of digoxigenin (DIG)-labeled antisense and sense RNA probes by transcription from FMO cDNA templates, the preparation of paraffin wax-embedded and cryostat tissue sections, the hybridization of RNA probes to tissue sections, and the specific detection of hybridized probes using an antibody to DIG.

In this chapter we describe the use of cRNA (riboprobes) in the detection of gene expression in tissue sections. Riboprobes offer good sensitivity and allow the detection of low-level mRNA expression. In some cases, the use of radiolabeling is justified because this method is still sensitive. However, recent advances in nonisotopic detection methods mean that in some cases digoxigenin (DIG) or biotin labeling also may be sufficiently sensitive to detect mRNA expression in tissues of interest. The use of alkaline phosphatase conjugated anti-DIG antibodies improves the sensitivity of DIG detection over peroxidase systems, and the use of amplification systems based on biotinyl tyramide has improved the sensitivity of biotin labelled probe detection. Finally, it can be shown that low-level mRNA expression is easier to detect in frozen sections than in paraffin-embedded material, with a consequent loss in quality of morphology.

Most vertebrate cells display a considerable microheterogeneity in their plasma membranes, often termed microdomain structure. Some of these microdomains are enriched in glycosphingolipids and cholesterol and are resistant to solubilization with nonionic detergents; they are therefore called detergent-insoluble-glycolipid enriched membrane (DIG) or glycosphingolipid enriched membrane (GEM). These domains, also called “lipid rafts” (Simons and Ikonen, 1997), may form at the plasma membrane (PM) upon external stimuli or may be present in a preassembled form upon vesicular traffic to and fusion with the PM (Simons and Ikonen, 1997; Brown and Rose, 1992). We consider lipid rafts as transient molecular associations between lipid and protein components of the PM, providing a dynamic patchiness and local order in the fluid mosaic membrane (Edidin, 2001). Although the microdomain concept is widely accepted, and the existence of rafts has been confirmed by many lines of experimental evidence (e.g., biochemical data on detergent resistance, resolving membrane patchiness by high-resolution fluorescence and electron microscopies, tracking by videomicroscopy the lipid and protein motions in the membrane, etc.), some basic questions about the microdomains still remain open or highly controversial.

In the following, we describe the application of the DIG/antiDIG system for the (structural) analysis of glycoproteins on blots. Special emphasis is being placed on the variety of the different DIG applications in order to obtain structural information concerning the glycoprotein carbohydrate chains. Prominent among them is the use of lectins with well-known specificities for carbohydrate structures.