Monoclonal antibodies fluorescent staining

It is entirely possible that surface staining cannot be accomplished before fixation. Some antibody-antigen complexes cannot withstand chemical fixation and/or permeabilization. An empirical evaluation must be made. In this example, cells are first stained with a monoclonal antibody against a cell-surface receptor, fixed with ethanol, and then the DNA is stained with propidium iodide. The cells are analyzed for two-color fluorescence, the green of the fluorescein-labeled surface marker and the red of the labeled DNA intercalator. This approach works for antibody-antigens that are unaffected by fixation. 

The actual response of monoclonal antibodies with individual cells is usually visualized either directly (typically using fluorescent stains) or indirectly [using the reaction of antibody labeled with horseradish peroxidase (HRP) or other enzymes] with diaminobenzidine (DAB) (or other substrate while using other enzymes) under the microscope or in the flow cytometer. The latter, however, is not employed routinely in CSF immunocytology, although it has an advantage in clinical hematology. 

Propidium iodide can be used to assess plasma membrane integrity in annexin V apoptosis assays. It does not cross the plasma membrane of cells that are viable or in the early stages of apoptosis because of their plasma membrane integrity. In contrast, cells in the late stages of apoptosis or already dead have lost plasma membrane integrity and are permeable to PI for DNA staining (Fig. 5). In flow cytometric assays, another nucleic acid dye that can be used in place of PI for the exclusion of nonviable cells is 7-AAD. The advantage of 7-AAD over PI is its ability to be used in conjunction with phycoerythrin (PE)- and FITC-labeled monoclonal antibodies with minimal spectral overlap between the 7-AAD, PE, and FTTC fluorescence emissions. 

The immunocytochemical staining of cytochrome C offers another alternative since, upon exposure to apoptotic stimuli, cytochrome C is rapidly released into the cytosol, an event that may be required for the completion of apoptosis in some systems (L2). The effect of cytosolic cytochrome C is thought to be the activation of caspases. The immunocytochemical staining of cytochrome C localized in mitochondria in healthy cells or diffused in the cell cytoplasm with monoclonal antibody (Promega) after induction of apoptosis, as detected by fluorescence microscopy, can be used for monitoring apoptosis (L2, M7, S8, T6). It is also a simple, rapid, specific mefhod for quanfifafive assessment of apoptotic cells. 

BrdU/DNA flow cytometry offers flexibility and diversity in the study of cell kinetics from cells in culture to human tumors in vivo. The essence of the procedure is to pulse label with BrdU by a short-term 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 then stained with a monoclonal antibody against BrdU that can be either directly conjugated to a fluoro-chrome (usually fluorescein isothiocyanate [FITC]) or, alternatively, bound to a second antibody conjugated with FITC. The cells are then counterstained with propidium iodide (PI) to measure the DNA content and analyzed on the flow cytometer. The results are displayed as linear-red fluorescence on the x-axis vs linear or log-green fluorescence on they-axis. 

To be of use in microscopy or flow cytometry, this bond needs to be visualized (to the eye or to the photodetector) by the addition of a fluorescent tag. Visualization can be accomplished by one of two different methods. With direct staining, cells are incubated with a monoclonal antibody that has been previously conjugated to a fluorochrome (for example, fluorescein or phycoerythrin or any fluorochrome with appropriate absorption and emission spectra). This procedure is quick and direct it merely involves a half-hour incubation of cells with antibody (at 4°C), followed by several washes to remove weakly or nonspecifically bound antibodies. Cells thus treated are ready for flow analysis (although final fixation with 1% electron microscopic-grade formaldehyde will provide a measure of biological safety and long-term stability). 

Fig. 6.17. The two-color fluorescence profile of peripheral blood mononuclear cells stained simultaneously with six different monoclonal antibodies to delineate five different populations of cells. From Floran et al. (1986).

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. 

Lymphocyte A lymphocyte is a particular type of white blood cell that is involved in many of an organism s immune responses. Subpopulations of lymphocytes with microscopically identical anatomy can be distinguished because their surface membranes contain different arrays of proteins. The staining of these proteins with fluorescently tagged monoclonal antibodies allows the subpopulations to be enumerated by flow cytometry. 

PBS is also commonly used as a wash buffer for IHC. PBS s advantages are reduced auto fluorescence in immunofluorescent assays, and it is relatively inexpensive compared to Tris-based buffers. However, in some cases PBS can cause higher levels of nonspecific staining, and it has been observed to reduce the specific binding abilities of certain monoclonal antibodies (Anti-CD30, for example). 

Direct fluorescent antibody smears have become a more efficient method than Giemsa stains or tissue cultures fiar identifying chlamydia. Commercially prepared kits make specimen collection convenient, and results are available in approximately 24 hours. Good results, however, depend on obtaining an adequate specimen. Fluorescein-labeled monoclonal antibodies in the staining reagent specific for Chlamydia trachomatis outer membrane proteins bind to the C. trachomatis in the smear. Studies that compare direct fluorescein antibody techniques with tissue culture results have found acceptable sensitivity and specificity values. 

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.]...

Fig. 2. Repeated injections of protein A deplete B-la cells in the PeC. Transgenic Xenomouse mice expressing human Igs [17] were injected intraperitoneally with 0 (open bars), 125 (gray bars), 250 (stippled bars) or 500 p,g (black bars) of recombinant protein A. After 24 h, peritoneal cells were washed and then cells were triple stained with fluorescent antibodies to human IgM (G20-127), mouse CD1 lb (Ml/70), and mouse CD5-PE (53-7.3). FACS analysis allowed identification of the B-la (IgM+CDl lb+CD5+) and B-lb cell subsets (IgM+CDllb+CD5—). All monoclonal antibodies and their corresponding isotype controls were purchased from BD PharMingen. p < 0.05. 

In immunofluorescence microscopy, specific proteins and organelles in fixed cells are stained with fluorescence-labeled monoclonal antibodies. Multiple proteins can be localized in the same sample by staining with antibodies labeled with different fluorochromes. 

Portion of a "lampbrush chromosome" from an oocyte of the newt Nophthalmus viridescens] hnRNP protein associated with nascent RNA transcripts fluoresces red after staining with a monoclonal antibody. [Courtesy... 

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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