Staining procedures, nucleic acids

Staining for thirty minutes in filtered 0.1% Alcian blue in 3% acetic acid, pH 2.5-2.8, colors the mucins of connective tissues, most epithelium, and certain microbial capsules (84h) deep blue to turquoise, with little or no staining of nucleic acids. Results with the Alcian blue stain resemble more those obtained with the better variants of the dialyzed iron procedure than those obtained with metachromatic dyes. In the case of epithelial mucins, Alcian blue has much greater sensitivity than toluidine blue. 

Several direct dye staining procedures can measure total protein biomass, nucleic acid content, or selective uptake by organelles such as mitochondria or lysosomes. Many staining methods are appropriate for small numbers of samples or for image-based high content screening but in most cases, the multistep procedures exhibit limitations for HTS. 

This same procedure can also be used in microscopy or FACS analysis coupled with a nuclear dye such as ethidium homodimer-1, a red fluorescent nucleic acid stain (ex/em 495/ 635 nm) that is only able to pass through the compromised membranes of dead cells, and indicates the proportion of live vs. dead cells. (The viability assay kit including calcein-AM and ethidium homodimer-1 is sold by Molecular Probes, Cat. L-3224.)... 

Taxonomic probes, including antibodies and nucleic acid or peptide nucleic acid (PNA) probes selectively stain particular target cells by association with antigens or DNA/RNA. The corresponding approaches are designated as immunofluorescence (IF) and fluorescence in situ hybridisation (FISH), respectively. The major limitation of these labelling procedures is the low fluorescence intensity, which results in poorly labelled cells that escape detection. 

The recognition of the limitations of short radiative fluorescence lifetime of some covalently bound labels used in studies of nucleic acids prompted Saavedra and Picozza to bind terbium to DNA via inunobilized DTPA [47]. Instead of the nanosecond lifetimes achieved with stains such as ethidium bromide, a radiative lifetime of 1.5 msec was obtained for terbium-labeled DNA. In addition, this material is stable under the conditions frequently encountered in polyacrylamide gel electrophoresis. This labeling technique could also be used for RNA. There can be no doubt that the requirements of genetic manipulation will bring about many similar procedures for analyzing nucleic acids. 

The cells are stained with a fluorochrome. The two most commonly used stains are 4, 6-diamidino-2-phenylindole (DAPl) [113] and acridine orange (AO) [63]. Both of those compounds bind nucleic acid molecules, DNA and RNA. A number of procedures have been developed for each one of dyes in question (e.g., stain, then filter versus filter... 

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