Fluorescence microscopy conjugates

The synthesis and characterization of a somatostatin receptor-specific peptide H2N-(DPhe)-cyclo[Cys-Phe-(D-Trp)-Lys-Thr-Cys]-Thr-OH, labeled with an indo-dicarbo- and an indotricarbocyanine dye at the V-terminal amino group were described in [34], The ability of these fluorescent contrast agents to target the somatostatin receptor was demonstrated by flow cytometry in vitro, wherein the indotricarbocyanine conjugate led to elevated cell-associated fluorescence on somatostatin receptor-expressing tumor cells. The intracellular localization was visualized using NIR fluorescence microscopy. 

Make it visible the fluorophore label can be visualized directly using fluorescent microscopy. The biotin label (see Sect. 6.2.1) can be detected using streptavidin conjugated with an enzyme the latter must be visualized through an enzyme chromogenic system. Incubate sections with an appropriate enzyme substrate until optimal color develops (see Sect. 2.3). 

For the design of mitochondriotropic liposomes, we have used a method, that has been a standard procedure in liposome technology for over 30 years the lipid-mediated anchoring of artificially hydrophobized water-soluble molecules into liposomal membranes (25-28). We have hydrophobized mitochondriotropic TPP cations by conjugating them to long alkyl residues specifically, we have synthesized stearyl TPP (STPP) salts (29). Following liposome preparation in the presence of STPP, the liposomal surface became covalently modified with TPP cations, thereby rendering these liposomes mitochondriotropic as verified in vitro by fluorescence microscopy (30). 

Giloh, H. and Sedat, M. (1982) Fluorescence microscopy reduced photobleaching of rhodamine and fluorescein protein conjugates by A-propyl gallate. Science 217, 1252-1255. 

Immunoassay kit, where the antibodies of 8-oxoguanine (96) are conjugated with fluorescein isothiocyante (97) as fluorophore and combine with the oxidized DNA. Detection of the greenish fluorescence is by fluorescence microscopy for tissues or by fluorescence-activated ceU sorting for cell suspensions . 

Figure 12.5 Nuclear import in permeabilized cells. HeLa cells were grown on coverslips and permeabilized with digitonin as described in Wilson et al., 1999. Fluorescein-PNA-labeled plasmids (containing the SV40 enhancer, 4.2 kb) or rhodamine-labeled BSA-NLS peptide conjugates were incubated with the cells for four hours at which time they were viewed by fluorescence microscopy. With no additions, neither DNA nor protein was imported, but in the presence of nuclear and cytoplasmic extracts both substrates localized to the nuclei. While plasmids containing the SV40 enhancer were taken up by the nuclei, those lacking the sequence were excluded. The remaining panels demonstrate the need for both the import machinery (importins and Ran) and a source of adapter proteins (nuclear extract) for plasmid nuclear entry, but not for protein nuclear localization.

Fig. 13.2. Preferred association of LCM with tumor cells in vivo. Vibratome sections of brain of rats bearing 9L tumor that have been injected i.v. with diO-LCM, and sacrificed 2 min later. Sections in panels A and C were stained with TR-conjugated WGA. Specimens in panels B and C were observed by fluorescence microscopy using fluorescein optics. The scale bar represents 100 pm. (Taken from ref. 531.)...

In keeping with current trends in immunohistochemistry to develop alternatives to biotin-streptavidin detection methods, a fluorescyl-tyramide amplification system has recently been introduced (FT-CSA). In this procedure peroxidase is associated with a tissue-bound primary antibody by application of a secondary antimouse Ig antibody to which peroxidase has been conjugated. The peroxidase catalyzes the conversion and deposition of fluorescyl-tyramide onto the tissue section. At this point the reaction can be terminated and viewed by fluorescence microscopy, or the signal can be converted to a colorimetric reaction by the sequential application of an anti-fluorsecein antibody conjugated to peroxidase followed by a diaminobenzidine-hydrogen peroxide substrate. 

The technique of immunohistochemistry is very similar to fluorescence microscopy. This technique differs only in the method of detection or localization of the antibody and can be performed with a conventional light microscope. As with the ELISA and Western blot, the antibody used in this experiment is covalently conjugated to an enzyme, such as horseradish peroxidase. This enzyme is then incubated with a substrate that is converted to an insoluble colored product that will precipitate or deposit at the site of enzyme activity. The distribution and location of the colored product is readily detected with an ordinary light microscope. 

The second step required in the application of avidin-biotin technology is to prepare an appropriate avidin-associated probe or probes for the desired application (for general reviews, see refs. 1 and 2). For example, a fluorescent form of avidin can be used for fluorescence microscopy, fluorescence-activated cell sorting, and in some cases, for immunoassay. Likewise, an avidin-enzyme conjugate can be used for immunoblotdng, immunoassay, light microscopy, and in some cases, electron microscopy. An immobilized form of avidin can be used for isoladon purposes (see Note 1). 

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