The Use of Fluorescent Probes in Immunochemistry*

Abstract

Abstract-The limitations and advantages of particular dyes for labelling proteins and other biological materials are discussed. Methods available for conjugating dyes to proteins are outlined. Following a discussion of double labelling methods the use of photoactivatable Ruorochromes and time resolved fluorescence methodologies are outlined. The reasons for the photoinstability of some fluorochromes are discussed and methods for overcoming the problem are described.

Flow Cytometric Applications of Sulforhodamine 101 as a Fluorescent Stain for Total Cellular Protein

“…A variety of other protein-binding dyes have also been tested for use as protein stains for flow cytometry (Freeman and Crissman 1975, Stohr et al 1977, 1978, Takahama and Kagaya 1988. Problems with FITC have included the fact that it is unstable in solution during storage and photobleaches upon irradiation (Stohr et al 1977, Davidson andHilchenbach 1990).…”

“…Since staining intensity is a function of both protein content and stain accessibility to the protein reaction sites, SR 101 fluorescence, with its ionic binding, could be a slightly different parameter t h a n FITC fluorescence [Dyson et al 1987). The monosulfonylchloride derivative of SR 10 1, which is commercially available as "Texas red," can be used to couple the dye to amino groups on proteins and other compounds, and has been used in conjunction with FITC in dual-color immunofluorescence studies (Titus et al 1982, Hardy et al 1982, Parks et al 1983, Davidson and Hilchenbach 1990.…”

Quantification of cell cycle kinetics by EdU (5-ethynyl-2′-deoxyuridine)-coupled-fluorescence-intensity analysis

“…Instead of screening for tagged mitotic fractions to identify cells that have left S phase other options involve pulsing replicating cells with two distinct nucleoside analogues at different times; or else, synchronizing the entire cell population to ensure an homogeneous entry in S phase and removal of the noise associated with double-pulsing methods [15]. Dual labelling requires the simultaneous use and detection of two antibodies specific for different analogues, hence special care needs to be taken to avoid cross hybridization signals [16]. Cell synchronization, on the other hand, carries the risk of disturbing normal cycle progression and inducing cell death, even when performed avoiding the use of drugs that target the cell cycle [17].…”

Following the dyeing of wool in real time using fluorescence microscopy

“…3 The ''noise'' displayed by the results is due to the fact that multiple positions were being monitored, necessitating moving the stage for each observation, and conse-quently readings were not always taken at exactly the same spot each time. [17][18][19] For Fig. This latter option has the disadvantage of being qualitative, but at the same time has the advantage of being able to record a number of fibers simultaneously and so gain an understanding of the presence/absence of certain structural features on the surface of the fibers.…”