Texas Red fluorescence

Record data from the samples labeled with all three antibodies. Display acytogramofgreen (CALLA) vs red (Texas Red) fluorescence. Set gates on the EMA and CALLA positive cells and display separate histograms of their orange c-erbB2) fluorescence. 

Fig. IV-19. Fluorescence micrographs showing the shape transitions in monolayers of dimyristoylphosphatidylcholine (DMPC) (84%) and dihydrocholesterol (15%) and a lipid containing the dye, Texas Red. (From Ref. 228.)...

Fig. 3 Polypeptide vesicle with endocytosis capability, (a) Vesicles formed from poly(L-arginme)6o-h-poly(L-leucme)2o- The poly(L-arginme) block provides an added cell-penetrating feature to the vesicles, (b, c) LCSM images of internalized vesicles (green) containing Texas-Red-labeled dextran (red) in (b) epithelial and (c) endothelial cells. Colocalization of the vesicles and Texas-Red-labeled dextran appears as a yellow fluorescent signal. Adapted from [44] with permission.Copyright 2007 Macmillan Publishers...

Since endocytosis ofLDH was confirmed by TEM images (Figure 13.9), forthe next step, its specific endocytic pathway for membrane entry was determined by immunofluorescence and confocal microscopy. Cells were incubated with LDH-FITC, fixed with 3.7% freshly made formaldehyde, and then stained with either anti-clathrin antibody or anti-caveolin-1 antibody both conjugated to the red fluorescent dye Texas Red (TR). The confocal microscopic images showed that green fluorescent... 

Four forms of amine-reactive rhodamine probes are commonly available. Two of them are based on the tetramethyl derivatives of the fundamental rhodamine structure, one is based on the sulforhodamine B or Lissamine derivative, and the last is the sulforhodamine 101 or Texas Red-type of derivative. All of them react under alkaline conditions with primary amines in proteins and other molecules to form stable, highly fluorescent complexes. 

Texas Red hydrazide is a derivative of Texas Red sulfonyl chloride made by reaction with hydrazine (Invitrogen). The result is a sulfonyl hydrazine group on the No. 5 carbon position of the lower-ring structure of sulforhodamine 101. The intense Texas Red fluorophore has a QY that is inherently higher than either the tetramethylrhodamine or Lissamine rhodamine B derivatives of the basic rhodamine molecule. Texas Red s luminescence is shifted maximally into the red region of the spectrum, and its emission peak only minimally overlaps with that of fluorescein. This makes derivatives of this fluorescent probe among the best choices of labels for use in double-staining techniques. 

Texas Red hydrazide is soluble in DMF and may be dissolved as a concentrated stock solution in this solvent prior to the addition of a small aliquot to an aqueous reaction medium. The solid and all solutions made from it must be protected from light to avoid photo-decomposition. Prepare the stock solution fresh immediately before use. A suggested protocol on the use of this fluorescent probe may be obtained by following the method outlined for fluorescein-5-thi-osemicarbazide in Section 1 of this chapter. Optimization may be necessary to achieve the best level of fluorescent modification (F/P ratio) for a particular application. 

In addition to the wide range of commercial probes, many other fluorescent molecules have been synthesized and described in the literature. Only a handful, however, are generally used to label antibody molecules. Perhaps the most common fluorescent tags with application to immunoglobulin assays are reflected in the main derivatives produced by the prominent antibody manufacturing companies. These include derivatives of cyanine dyes, fluorescein, rhod-amine, Texas red, aminomethylcoumarin (AMCA), and phycoerythrin. Figure 20.16 shows the reaction of fluorescein isothiocyanate (FITC), one of the most common fluorescent probes, with an antibody molecule. 

Titus, J.A., Haugland, R.P., Sharrow, D.M., and Segal, J. (1982) Texas Red, a hydrophilic, red-emitting fluorophore for use with fluorescein in dual parameter flow microfluorometric and fluorescence microscopic studies./. Immunol. Meth. 50, 193-204. 

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. 

An immunosensor based on a competitive fluorescence energy-transfer immunoassay was reported by Anderson 105) for the measurement of phenytoin. Texas red-labeled antibody was incubated with a phenytoin derivative. On displacement of the derivative by the antigen, the change in the fluorescence signal was recorded. Detection limits approached 5 /iM with response times ranging from 5 to 30 min. 

Fluorescence resonance energy transfer has also been used for ionic strength measurements.(95) Fluorescein labeled dextran (donor) and polyethyleneimine-Texas Red (acceptor) were placed behind a dialysis membrane. The polymer association is ionic strength dependent and the ratio of intensities (F o/Fw) was used as the measured parameter. Since both the donor and acceptor are fluorescent, this kind of sensor may allow expand the sensitive ionic strength range by shifts in observation wavelength, as was discussed for pH probe Carboxy SNAFL-2 (see Section 10.3). 

Figure 14.18. Diagrammatic representation of a phenytoin fluorescence energy transfer fiber optic sensor, as described in the text. -< = Antibody-Texas Red P = unlabeled phenytoin P = phenytoin-b-phyco-erythrin. (Adapted from Ref, 119.)...

P. Suci and V. Hlady, Fluorescence lifetime components of Texas Red-labeled bovine serum albumin Comparison of bulk and adsorbed states, Colloids Surf. 51, 89-104 (1990). 

The fluorescent labels reported for investigation of intracellular uptake and distribution by CLSM comprise Nile red [13], Texas Red, and 6-coumarin [14]. Not only for fluorescence microscopy but also for transmission electron microscopy (TEM), the loading of markers proved to be useful. Osmium tetroxid as an electron dense marker and bovine serum albumin (BSA) as a model protein were entrapped in PLGA-nanoparticles to elucidate their uptake and intracellular distribution in human vascular smooth muscle cells [15]. 

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). 

Fluorescence detection relies on the visualization of a secondary antibody that has been labeled with a fluorophore such as fluorescein (FITC), Texas Red, Tetramethyl rhodamine (TRITC), or R-phycoerythrin. Although this method of detection has a reduced sensitivity of twofold to fourfold compared to chemiluminescence detection, it presents a tenfold greater linear dynamic range, thus providing better linearity and better quantiflcation within the detection limits. Since secondary antibodies can be labeled with fluor-ophores of distinct colors, multiplexing (simultaneous detection of several antigens) of the same blot is feasible. 

Fig. 3. Fluorescence profiles of 2, 7 -dichlorofluorescin-loaded cells assayed in whole blood. (A) Compares the fluorescence histograms of unstimulated, control cells (shaded curve) with granulocytes exposed to opsonized S. aureus (open curve). (B) illustrates the two-color analysis profde of the granulocytes that were exposed to Texas Red-labeled S. aureus. Red fluorescence is the result of particle association with each granulocyte, whereas green fluorescence is the result of the oxidation of 2, 7 -dichlorofluorescin to 2, 7 -dichlorofluorescein (DCF). The red and green fluorescence analyses were performed with log-scale detection amplification for each fluorochrome.

Wang et al. injected a Texas-red-labelled phosphorothioated AS-ODN into the dopamine lA receptor in the rat renal interstitium. Fluorescence was detected after 24 h in both tubular epithelium and intra-renal vasculature. Treatment resulted in a 35% decrease in the dopamine lA receptor protein, causing a reduction in urinary sodium excretion and urine output [131],... 

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