Fluorescence staining

The presence of mycoplasmal DNA in the cell cytoplasm or attached to the cell membrane may be detected by staining with the fluoro-chrome Hoechst 33258 (Appendix 3). This intercalating dye fluoresces under ultraviolet light and this forms the basis of a very rapid test for mycoplasmas. 

The fluorescence consists of a diffuse series of small granules apparently throughout the cytoplasm of infected cells. Confusion may arise from much larger, brightly staining fragments of nuclei which sometimes contaminate a culture (McGarrity et al., 1983). 

A stock of the Hoechst 33258 bisbenzamid fluorochrome solution is made by dissolving 5 mg in 100 ml PBS-A (Appendix 1) using a magnetic stirrer. It must be free of bacterial contamination and should be sterilised by filtration through a 0.22 (im membrane and should be stored in the dark at 4°C. It should be diluted 100-fold with filtered PBS-A for use. 

Russel et al. (1975) report a similar staining method using 4, 6-di-amidino-2-phenylindole (DAPI)  

Autoradiography and staining methods are based on detection of cytoplasmic DNA (in the former case replicating cytoplasmic DNA). For this reason there will always be a low background caused by mitochondrial DNA and samples to be tested should be compared with positive and negative controls. Such prefixed controls are available from Moredun Ltd. (Appendix 3) ready for staining. 

In vitro staining is used to colour cells or structures that have been removed from their biological context. Certain stains are often combined to reveal more details and features than a single stain alone can provide. Combined with specific protocols for fixation, sample preparation and investigative methods such as fluorescence microscopy, these standard staining techniques are used as consistent, repeatable diagnostic tools. 

Panel (d) shows magnification of proliferated cells with extended filopodia. Ethidium bromide/acridine orange combined stain causes living cells to fluoresce green whereas apoptotic (dead) cells cause a distinctive red-orange fluorescence (Amaravathy et al., 2014). ( With permission by Elsevier.) 

Melero et al. (2014) studied the morphology, proliferation and attachment of human osteoblastic cells at the surfaces of HVOF-sprayed hydroxyapatite/titania 

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Ostle, A., Holt, J.G., 1982. Nile blue A as a fluorescent stain for poly-b-hydroxybutyrate. 

Szewczyk B, Summers DF (1987) Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera. Anal Biochem 164 303-306... 

Vassar PS, Culling CF (1959) Fluorescent stains, with special reference to amyloid and connective tissues. Arch Pathol 68 487... 

During the past two decades, cell-biological and biomedical research has greatly benefited from innovations in fluorescence microscopy. Both the increase in the repertoire of fluorescence staining techniques at the (sub)cellular level and the development of a multitude of novel fluorescence microscopy techniques contributed significantly. 

Fluorescent stains such as DAPI and ethidium bromide bind to DNA (sometimes even mitochondrial DNA) and have many uses, including location of nuclei and rapid detection of microorganisms or virus formation (22). The lipophilic stain DiOC(6) can be used to stain mitochondria and endoplasmic reticulum in living cells (23,24), and is also a potential-sensitive stain. 

Suberized lamellae in casparian strips can be located using the berbe-rine-aniline blue fluorescence staining method, which works equally... 

Brundrett MC, Enstone DE, Peterson CA. A herherine-aniline blue fluorescent staining procedure for suberin, lignin and callose in plant tissues. Protoplasma 1988 146 133-142. 

Mitochondria are distinct organelles with two membranes. The outer membrane limits the organelle and the inner membrane is thrown into folds or shelves that project inward and are called cristae mitochondriales. The uptake of most mitochondrion-selective dyes is dependent on the mitochondrial membrane potential. Conventional fluorescent stains for mitochondria, such as rhodamine and tetramethylrosamine, are readily sequestered by functioning mitochondria. They are, however, subsequently washed out of the cells once the mitochondrion s membrane potential is lost. This characteristic limits their use in experiments in which cells must be treated with aldehyde-based fixatives or other agents that affect the energetic state of the mitochondria. To overcome this limitation, the research... 

Precise quantifications are an important quality in molecular biology. There are slight differences in the methods used for global and targeted proteomics. In experiments intended to visualize as many proteins as possible, it is highly desirable to have a parallel quantification method that builds on the display technique. For 2D gel electrophoresis, fluorescent staining methods are under development (Urwin and Jackson, 1993), but they still lack overall sensitivity. Labeling proteins with radioactive isotopes is the most precise method for quantification but is limited to cell cultures, and alternatives are desirable. Recently, a precise method... 

Huang, S. N., Minassian, H., and Moore, J. D. (1976) Application of immuno-fluorescent staining on paraffin sections improved by trypsin digestion. Lab Invest. 35, 383-390. 

Fluorescence staining for flow cytometric analysis falls into three categories methods in which a fluorescent ligand accumulates on or within the cell (see Chapters 36,38) methods that require the ligand to interact with a cellular component to release the fluorophore or result in light emission (see Chapters 34,39) and methods that rely on fluorophore-coupled antibody binding (see Chapters 32,33). 

Battaglia, C., Salani, G., Consolandi, C., Bemardi, L.R., and DeBellis, G., Analysis of DNA microarrays by non-destmctive fluorescent staining using SYBR green 11, Biotechniques, 29(1), 78-81, 2000. 

The actual response of monoclonal antibodies with individual cells is usually visualized either directly (typically using fluorescent stains) or indirectly [using the reaction of antibody labeled with horseradish peroxidase (HRP) or other enzymes] with diaminobenzidine (DAB) (or other substrate while using other enzymes) under the microscope or in the flow cytometer. The latter, however, is not employed routinely in CSF immunocytology, although it has an advantage in clinical hematology. 

Fluorophore stains Fluorescent detection of proteins following electrophoresis during large-scale proteome analysis is achieved by fluorophore stains. Their detection sensitivity is superior to colorimetric stains. The most commonly used fluorescent stains are... 

Nishihara, J.C., Champion, K.M. (2002). Quantitative evaluation of proteins in one- and two-dimensional polyacrylamide gels using a fluorescent stain. Electrophoresis, 23(14), 2203-2215. 

Martin, F.W. 1959. Staining and observing pollen tubes in the style by means of fluorescence. Stain Technol. 34 125-128. 

The search for more rapid and sensitive methods of protein detection after electrophoresis led to the development of fluorescent staining techniques. Two commonly used fluorescent reagents are fluorescamine and anilinonaphthalene sulfonate. New dyes based on silver salts (silver diamine or silver-tungstosilicic acid complex) have been developed for protein staining. They are 10 to 100 times more sensitive than Coomassie Blue (Fig. 4.7). 

Sensitivity of fluorescent stains is very dependent on protein amino acid composition. Minimum amount detected based on amount of protein loaded onto gel. The actual amount on the blot will be slightly lower because of losses during electrotransfer. Values are based on use of a full-sized gel (I I cmx I6cmx 1.5 mm). Sensitivity will be -2 to 5 times higher when minigels (8 cm x 10 cm x 1.0 mm) are used because the protein bands are concentrated on a smaller area of membrane. 

FITC-Bac) delivered in vivo from ethosomes, penetrated the rat skin through the intercor-neocyte pathways, which typically exist along the lipid domain of the stratum corneum [95] (Figure 13.7). In contrast, significantly lower fluorescence staining of the intercellular penetration pathway and no inter- or intracorneocyte fluorescence were observed with FITC-Bac hydroethanolic solution and liposomes, respectively. 

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