Antibodies conjugation with fluorochromes

The fluorochromes, Calcofluor White and Congo red bind to chitin and cellulose, while acridine orange binds to nucleic acids of the fungi. These dyes may be helpful when there is a lack of fungal elements (blastospores, hyphae or pseudohyphae) in the specimen, as even few elements are visible under the fluorescence microscope. By using specific antibodies conjugated with the fluorochromes, it should be possible to identify specific fungi. 

The essence of the procedure is to pulse label with BrdUrd by a short incubation in vitro or by a single injection in vivo, samples are then taken at time intervals thereafter, and stained after fixation in ethanol. The cells are stained using a monoclonal antibody against BrdUrd that can either be directly conjugated to a fluorochrome (usually FITC) or alternatively bound to a second antibody conjugated with FITC. The cells are then counterstained with PI to measure DNA content, and analysed on the flow cytometer for red (DNA) and green (BrdUrd) fluorescence. The results are displayed as red (x axis) versus green y axis) bivariate distributions. 

Species-specific secondary antibodies (1 500) conjugated with fluorochromes at different excitation wavelengths depending on the microscope (e.g., 488 nm for green or 594 nm for red fluorescence). Sec Note 5. 

Goding, J.W. (1976) Conjugation of antibodies with fluorochromes Modifications to the standard methods./. Immunol. Meth. 13, 215-226. 

Fluorochromes are stains that interact directly with cellular components, or are used to form conjugates with antibodies or ligands, yielding fluorescent reporter molecules. O Table 13-1 lists a selection of fluoro- D Table 13-1 Fluorochromes for flow cytometry ... 

Robins, R A, Laxton, R R., Garnett, M., Price, M. R., and Baldwin, R W (1986) Quantitation of antitumor antibody and antibody conjugate binding activity by competition with fluorochrome-labelled antibody J Immunol Methods 90, 165—172. 

When one or more of the desired antibodies is not available as a direct conjugate and a conjugate cannot be synthesized, indirect methods have to be used. The simplest variation is to use a biotin-labeled antibody as a first layer, and then after washing the cells, incubate with fluorochrome-labeled streptavidin, and additional directly labeled antibodies labeled with complementary fluoro-chromes. The additional streptavidin step does not usually cause difficulty... 

Isotype-matched unconjugated, biotin-conjugated, or fluorochrome-labeled antibodies unreactive with mammalian cells are available as control reagents from many suppliers... 

Although many applications of flow cytometry involve the staining of cells for proteins expressed on the outer membrane, cells also have many proteins that are not displayed on their surface. With appropriate procedures, flow cytometry can provide a means to analyze these intracellular proteins. The outer cell membrane is impermeable to large molcules like antibodies however, if we intentionally fix cells to stabilize proteins and then disrupt the outer membrane, the cells can be stained with fluorochrome-conjugated monoclonal antibodies against intracellular proteins. After time to allow the antibodies to pass through the now-permeabilized membrane, the cells are washed to remove loosely bound antibodies and then are run through the flow cytometer to measure their fluorescence intensity. 

Preparations are incubated with appropriate reagents to allow visualization based upon the detection system associated with the secondary antibody. The secondary antibody may be conjugated to a enzyme (e.g., alkaline phosphatase, horseradish peroxidase). Incubation with the appropriate substrate to the enzyme will result in the production of an insoluble colored product that can be detected upon microscopic analyses of the cells. Secondary antibodies can also be conjugated to fluorochromes (e.g., fluorescein, rhodamine) that can be detected using a microscope equipped to detect fluorescence. Immunohisto-chemistry has proven to be a powerful tool in biochemical toxicology allowing for in situ assessments of protein responses to toxicant exposure. 

Fluorochrome labeling of streptavidin or antibody Conjugation procedures should yield optimal fluorochrome/protein (F/P) ratios. Most economically, the desired F/P ratio is regulated by the initial weight of dye in the reaction mixture and the reaction is allowed to go to completion. Alternatively, with relatively more dye, the reaction is interrupted after a specific incubation period. The efficiency of labeling depends on the protein, the protein concentration, the specific fluorochrome and the purity of the fluorochrome (some preparations only 30%). Over- or undercoupling leads to nonspecificity or low detectability, respectively. 

Fluorescence-based immunolocalization of biomolecules within cells and tissues requires that fluorochromes, or fluorescent markers such as quantum dots, be covalently conjugated to antibodies. Techniques for antibody-fluorochrome conjugation were first devised by A. H. Coons and his associates, who pioneered the use of immunofluorescence microscopy in the 1940s and 1950s. Initially, the conjugation of fluorochrome to antibody was done directly to the antibody with the desired antigenic specificity (1). Subsequently, an indirect method (2) of immunolocalization was introduced that proved advantageous for most routine work. 

Secondary antibody (usually antimouse conjugated with a fluorochrome). 

Incubate the sections in the secondary antibody conjugated to the desired fluorochrome (see Note 47) and proceed with standard immunohistochemical procedures. Fig. 2B demonstrates the appearance of an immunohistochemically stained lacZ-positive cell. 

Stain the slides with second antibody conjugated to a suitable fluorochrome, as in... 

Fig. 5 Vertical distribution of the fibrillar fibronectin network (stained with fluorochrome conjugated antibodies) aroiuid endothelial cells (stained with CellTrackerGreen (Invit-rogen)) after 5 days of cell cultiue on two polymer substrates with different anchorage strength [left—poly(octadecene-a/t-maleic anhydride), right—poly(ethylene-a/f-maleic anhydride)]. The insets show the lateral network pattern scale ban 100 xm). Visualization and analysis was performed on a confocal laser scanning microscope (TCS SPl, Leica)...

A number of molecular biology procedures have also been developed to detect in situ some DNA modifications primarily related to apoptosis and programmed cell death (PCD). These procedures can be collectively referred as in situ end-labeling (ISEL) techniques and are primarily based on the incorporation of digoxigeninated nucleotides into DNA that are subsequently visualized by anti-digoxigenin antibodies that can be conjugated with AP or a fluorochrome [113],... 

---