Conjugation of fluorochromes to antibodies.

Immunolocalization of antigen via fluorescence requires that fluorochromes be linked either to the primary antibody (direct method) or to a second antibody (indirect method) to provide a fluorescent signal to mark the site of antibody-antigen binding. Of these two methods, the indirect technique is generally more useful and practical. Fluorochromes can be covalently conjugated to antibodies through reactions with thiol or amine groups. Typically, fluorochromes containing isothiocyanate, succinimidyl ester, or sulfonyl chloride reactive groups are conjugated to amines on the antibody molecules. Provided are step-by-step instructions for conjugating isothiocyanate derivates of fluorescein and sulfonyl chloride derivatives of rhodamine to the amine groups of antibodies.

Fluorochromes: properties and characteristics.

Immunofluorescence microscopy provides a sensitive means by which antigens can be localized within tissues or individual cells. For the most effective use of this technique the researcher can draw upon basic information on factors that affect the brightness of the fluorescence image, and how well that image can be distinguished from background fluorescence or interfering fluorescence signals. A wide variety of fluorochromes are available, with emitting wavelengths that range from the blue-violet end of the visible spectrum to the infrared. Individual fluorochromes are characterized by their extinction coefficients, quantum yields, susceptibility to photobleaching, the wavelengths at which they maximally absorb excitatory and emit fluorescent light, and how far apart those wavelength maxima are separated. Additional choices for fluorescent labeling of antibodies are provided by the availability of fluorescent quantum dots. Informed choices of fluorochromes can obviate many problems, particularly with regard to situations in which two or more antigens are to be localized simultaneously within a specimen.

Overview of conventional fluorescence photomicrography.

In an age of digital imaging, photographic film still provides a viable and effective means for recording fluorescence images by photomicrography. To maximize the quality of results that are obtained, a photographic emulsion with sufficient sensitivity for the low light level characteristic of Immunofluorescence must be selected, exposures adjusted for reciprocity failure, and modern, high numerical aperture objective lenses employed to produce the brightest possible image. Mounting media that reduce the effects of photobleaching on fluorochromes also help to maintain image brightness, and so reduce exposure times. Digital scanning of film-based micrographs provides the convenience of utilizing image processing software to adjust image density and contrast, and to produce quality prints.

Pdr5-mediated multidrug resistance requires the CPY-vacuolar sorting protein Vps3: are xenobiotic compounds routed from the vacuole to plasma membrane transporters for efflux?

In Saccharomyces cerevisiae several members of the ATP-binding cassette transporter superfamily efflux a broad range of xenobiotic substrates from cells. The vacuole also plays a critical role in multidrug resistance. Mutations in genes such as VPS3 that are essential for vacuolar acidification and carboxypeptidase Y vacuolar protein-sorting are multidrug sensitive. A similar phenotype is also observed with deletions of VPS15, VPS34, and VPS38, which encode essential members of the carboxypeptidase Y vacuolar protein-sorting pathway. Prior to the work described herein, detoxification by transporters and the vacuole were presumed to function independently. We demonstrate that this is not the case. Significantly, Vps3 has an epistatic relationship with Pdr5, a major yeast multidrug transporter. Thus, a double pdr5, vps3 deletion mutant is no more multidrug sensitive than its isogenic single-mutant counterparts. Subcellular fractionation experiments and analysis of purified plasma membrane vesicles indicate, however, that a vps3 mutation does not affect the membrane-localization or ATPase activity of Pdr5 even though rhodamine 6G efflux is reduced significantly. This suggests that Vps3 and probably other members of the carboxypeptidase Y vacuolar protein-sorting pathway are required for relaying xenobiotic compounds to transporters in the membrane.