Fluorescent labeling fluorescein

Calibration. Many approaches have been used to calibrate flow cytometric measurements. Including the comparison of flow and nonflow techniques (radiolabels, spectrofluorometry). In recent years, commercial standards have been introduced which are calibrated in fluorescein equivalents/particle (e.g., 3,000 or 500,000). With labeled ligands, calibration requires determining the relative quantum yield of the ligand compared to pure fluorescein and using the standards to analyze the amount bound on cells. Our ligands (fluorescein isothiocyanate derivatives) are typically 50% as fluorescent as fluorescein. 

The stoichiometry of the recharged DNA/PLL/SPLL particles was studied using sucrose-gradient ultracentrifugation of fluorescently labeled polyion complexes in 25 mM HEPES buffer. Rhodamine-labeled DNA (Rh-DNA) and either fluorescein-labeled PLL (Fl-PLL) or SPLL (Fl-SPLL) were used to determine their relative amounts within DNA... 

The nondestructive introduction of a fluorescent label would provide the molecule with a nonradioactive fluorophore, yet would preserve the option for direct radiolabelling of the fluorescent moiety with 125Iodine. This approach was pioneered by Nagasawa et al. (5) who reacted native or /V-desulfated heparins with a fluorescein isothiocyanate (FITC). The resulting degree of labelling was low... 

One of the first practical applications for these fluorescent labelled heparins was to examine the heparin binding behavior of different proteins and peptides under study in our laboratories. To this end we used a modification of the dot-blot assay described by Hirose and colleagues (13). F-D labelled heparin (-1 fluorescein/heparin) was radiolabelled with 125Iodine using iodobeads, to a specific activity of approximately 0.5 x 106 cpm/pg. Solutions of proteins with known heparin-binding capacities were dotted on nitrocellulose paper. A series of replicates... 

Elimination from the vitreous occurs by one of two pathways. This can be visualized by injecting fluorescent compounds and examining the concentration distribution in frozen sections obtained after a steady state has been established [230]. If the major route of elimination is by means of the re-tina/choroid, at steady state the lowest concentration would be in the vicinity of the retina. The contours observed in frozen sections of the rabbit eye obtained after intravitreal injection of fluorescein exhibit this pattern, with the highest concentration immediately behind the lens (Fig. 16A). Compounds not chiefly eliminated through the retina exit the vitreous by passive diffusion and enter the posterior aqueous, where they are eliminated by the natural production and outflow of aqueous humor. In such a situation, the contours would be perpendicular to the retina, with the highest concentration towards the rear of the vitreous cavity. This appears to be the case for fluorescently labeled dextran polymer, whose contours decrease in concentration toward the hyaloid membrane (Fig. 16B). 

Fluorescein and its derivatives represent one of the most popular of all fluorescent labeling agents. Its fluorescent character is created by the presence of a multi-ring aromatic structure due to the planer nature of its upper, fused three-ring system (Figure 9.4). In its most elementary... 

TRITC has been used in numerous applications involving fluorescence detection, including double-staining techniques with fluorescein-labeled probes (Mossberg and Ericsson, 1990), the synthesis of fluorescently labeled DNA probes (Smith et al., 1985), as a label in homogeneous... 

The spectral properties of four major phycobiliproteins used as fluorescent labels can be found in Tables 9.1 and 9.2. The bilin content of these proteins ranges from a low of four prosthetic groups in C-phycocyanin to the 34 groups of B- and R-phycoerythrin. Phycoerythrin derivatives, therefore, can be used to create the most intensely fluorescent probes possible using these proteins. The fluorescent yield of the most luminescent phycobiliprotein molecule is equivalent to about 30 fluoresceins or 100 rhodamine molecules. Streptavidin-phycoerythrin conjugates, for example, have been used to detect as little as 100 biotinylated antibodies bound to receptor proteins per cell (Zola et al., 1990). 

DNA modified with a diamine compound to contain terminal primary amines may be coupled with amine-reactive fluorescent labels. The most common fluorophores used for oligonucleotide labeling are the cyanine dyes and derivatives of fluorescein and rhodamine (Chapter 9). However, any of the amine-reactive labels discussed throughout Chapter 9 are valid candidates for DNA applications. 

Albert H. Coons (Fig. 1.2) was the first who attached a fluorescent dye (fluorescein isocyanate) to an antibody and used this antibody to localize its respective antigen in a tissue section. The concept of putting a visible label on an antibody molecule appeared both bold and original. His initial results were described in two brief papers in the early 1940s (Coons et al. 1941,1942), but the research was halted while he joined the army and spent the next 4 years in the South Pacific. His later studies (Coons and Kaplan 1950) contributed immensely to the use of the fluorescent antibody method in a wide variety of experimental settings. In our time, the use of antibodies to detect and localize individual or multiple antigens in situ has developed into a powerful research tool in almost every field of biomedical research (http //books.nap.edu/html/biomems/acoons.pdf). 

Albert H. Coons was the first to attach a fluorescent dye (fluorescein isocyanate) to an antibody and to use this antibody to localize its respective antigen in a tissue section. Fluorescein, one of the most popular fluorochromes ever designed, has enjoyed extensive application in immunofluorescence labeling. For many years, classical fluorescent probes such as FITC or Texas red (TR) have been successfully utilized in fluorescence microscopy. In recent decades, brighter and more stable fluorochromes have continually been developed (see Table 14.1). Marketed by a number of distributors, cyanine dyes, Cy2, Cy3, Cy5, Cy7, feature enhanced water solubility and photostability as well as a higher fluorescence emission intensity as compared to many of the traditional dyes, such as FITC or TR. 

FIA Fluorescence immunoassay uses a fluorescent tag on the antibody or antigen. Fluorescent labels absorb light of one wavelength and reemit it at another wavelength. The label is excited by UV and emits visible light. Common fluorescent labels are fluorescein, Texas red, and GFP (green fluorescent protein). 

Fig. 2. Apoptotic cells fluorescently labeled using the TUNEL assay. Fixed paraffin-embedded rat mammary glands, 4 d postweaning (obtained from Oncor), were labeled using the TUNEL assay to detect apoptotic cells. Reagents were from Oncor s Apoptag Direct In Situ Apoptosis Detection Kit (Fluorescein), which directly incorporates fluorescein-labeled dUTP in the TdT end-labeling reaction. All reagents were used as described by the manufacturer (see Note 1). (Abbreviations are as in text.)...

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