Fluorescent labelling of antibodies

The antibodies used for conjugation to fluorescent dyes should be purified and highly specific since contaminating molecules or inactive antibodies in the labelling solution will also be fluorescently labelled and may bind to the sample in a non-specific way causing a disturbing background fluorescence. 

Several bright and photostable fluorescent dye derivatives, which are reactive with free amine or sulfhydryl groups (as found for example in lysines or cysteines, respectively) are available. Typical dye derivatives used for the labelling of amines or sulfhydryls are the isothiocyanates and carboxy-sucdnimidyl esters or iodoacetamides and maleimides, respectively. Amine containing side-chains are usually localized on the surface of a protein and thus they are very 

Some amine-specific dye derivatives and their excitation and emission maxima are listed in Table 1. 

Below we describe a protocol for the fluorescent conjugation of antibcxlies which we routinely use in our laboratories. A more general discussion of fluorescent dye derivatives and their applications to the labelling of marker proteins can be found in ref l. 

Labelling of antibodies with tetramethylrhodamine-S-isothiocyanate (TRITC) 

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Fluorescent Labeling of Antibodies Several Cyanine Dyes are Commercially Available 1. Dimethylsulfoxide (DMSO). 2. Dissolving buffer. 100 mM NaCl, 50 mM NaH PO, 1 mM EDTA, pH adjusted to 7.5 with NaOH. 3. 100 mM NaH PO. 4. PD-10 desalting columns. 

This chapter discusses protocols for fluorescent labelling of antibodies, optimized fixation, and permeabilization of cells, and describes several immunostain-ing procedures with the aim to quantitatively anafyse the localization of antigens in fixed and living cells. Various aspects of the imaging equipment presently available, and the ways how to most efficiently use it in order to quantify and document results will be discussed. Labelling in living cells by microinjection of fluorescently labelled antibodies and subsequent time-lapse microscopy will also be discussed. 

Fluorescent labelling of antibodies 356 Fixation, penneabilization, and staining of cells 358... 

In some cases, the preparation of a fluorescently labeled antibody is not even necessary. Particularly, if indirect methods are used to detect antibody binding to antigen, then preparing a fluorescently labeled primary antibody is not needed. Instead, the selection from a commercial source of a labeled secondary antibody having specificity for the species and class of primary antibody to be used is all that is required. However, if the primary antibody needs to be labeled and it is not manufactured commercially, then a custom labeling procedure will have to be done. 

There are literally hundreds of markers that are currently available for the mouse and human than can be used to characterize lymphoid and myeloid cells and subsets in primary and secondary lymphoid organs. Many of the markers expressed in mammals are highly conserved across species and have been designated as genetic clusters of differentiation (CD). CDs can be identified with fluorescently labeled monoclonal antibodies. As presented previously, when combined with other fluorescent probes, important information on intracellular biochemistry and cell function can be obtained. Many of the biochemical markers used by immunotoxicologists are common to other... 

Fluorescence immunoassays require labeling of antibodies, antigens, or both. The structure and some of the properties of antibodies that are important to the construction of immunoassays are briefly discussed first in this section, followed by a general discussion of probes and some of their characteristics. 

In case of fluorescent labelled secondary antibody mount the slide and visualise under fluorescent or Confocal microscope. For peroxide visualisation incubate with a DAB solution till brown color is seen followed by stopping the reaction under running tap water. 

Ericsson, 1990), the synthesis of fluorescently labeled DNA probes (L. M. Smith et al., 19 8 5), as a label in homogeneous immunoassay systems (Nithipatikom and McGown, 1987), to investigate specific interactions of proteins with cells surfaces (Hochman et al., 1988), and as an important fluorescent tag of antibodies in immunohistochemical staining techniques (Davidson and Hilchenbach, 1990). 

Fluorescence Scan of an Array of 1024 Peptides in A 1.6-cm2 Area. Each synthesis site is a 400- am square. A fluorescently labeled monoclonal antibody was added to the array to identify peptides that are recognized. The height and color of each square denote the fluorescence intensity. [After S. P. A. Fodor, J. O. Read, M. C. Pirrung, L. Stryer, A. T. Lu, and D. Solas. Science 251(1991) 767.]... 

Figure 3.31 Fluorescence micrograph of a developing Drosophila embryo. The embryo was stained with a fluorescence-labeled monoclonal antibody for the DNA-binding protein encoded by engrailed, an essential gene In specifying the body plan. [Courtesy of Dr, Nipam Patel and Dr, Corey Goodman.]...

A EXPERIMENTAL FIGURE 20-35 Cytosolic dynein participates in the formation and stabilization of mitotic spindle poles. In vitro reconstituted spindles were stained with fluorescently-labeled polyclonal antibodies to tubulin (green) and dynein (red) and examined with a fluorescence microsope. In the control spindle left), cytosolic dynein is present at each tapered end, as well as at the equator. Addition of a dynein monoclonal antibody after the formation of the spindle right) disrupts dynein localization and causes the poles to splay. [From R. Fleald et at, 1997, J. Cell Biol. 138 615 courtesy of R. Fleald.]... 

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