Antibodies direct staining with

Fig. 3. Calibration of phycoerythrin (PE) staining using Simply Cellular Beads, Bead antibody binding capacity was plotted against the corresponding bead s channel position, after (A) direct staining with PE-antireceptor mouse MAb, and (B) after staining with mouse MAb and PE-antimouse IgG. The higher sensitivity of the indirect method is reflected in the rightward shift of the calibration curve.

Fig. 3. Three-color analysis of peripheral blood lymphocytes for CD45RA, CD45RO, and CD4. Lymphocytes were stained with antibodies directly labeled with FITC, PE, and PECy5, respectively. The distribution of control labeled cells is shown in A and B, and labeled cells in C—F. The relationship between CD45RA and CD45RO in CD4-positive cells is shown in (F), which is gated for PECy5-positive cells.

Whereas multicolor immunoenzyme staining is applicable only for separately located antigens (see Chap. 7), multicolor fluorescence immmunostaining makes it possible to colocalize antigens not only in the same cell but also in the same cellular compartment. Simultaneous immunolocalization of antigens using fluorescent antibodies can be fulfilled both by the direct (see Sect. 4.1) and indirect (see Sect. 4.2) methods. With the direct method, primary antibodies are labeled with fluorescent dyes, while with the indirect method, primary antibodies are applied as unlabeled antibodies and the visualization is performed with secondary antibodies that are labeled with fluorescent dyes. 

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. 

Plot the peak positions against the manufacturer s specified antibody binding capacity for each bead in the mixture to create a standard curve. Figure 3 illustrates curves for Simply Cellular beads stained with a PE-labeled antibody in a direct assay (A), and m an indirect assay with a PE-labeled antimouse IgG (B). The increased sensitivity of the indirect assay is evident from the rightward shift of the curve. 

Analysis of samples of whole blood stained with directly labeled antibodies (Section 3.1.) will be used to illustrate cytometric analysis of multiple fluoro-chromes. 

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). 

Sometimes tissues are frozen and then sectioned so that they can be cut directly without the need for embedding. In other instances tissues are embedded in a polymer such as methacrylate instead of paraffin after fixation. There are several variations that are used to make tissue sections to view under the light microscope. In addition to staining with dyes, fluorescent molecules can be specifically attached to macromolecules using antibodies. 

Simultaneous staining In a simultaneous double stain, the primary antibodies can be applied simultaneously. The advantage of this method is that it is less time-consuming because the reagents can be mixed together. However, the technique can only be used, if suitable primary antibodies are available. Two methods can be adopted A direct method with directly-labeled primary antibodies, or an indirect method based on unlabeled primary antibodies raised in different host species, or of different Ig isotype or IgG subclass (4). 

Figure 11.8. Detection of NOS-dependent S-nitrosylation in vivo, a Immunochemical detection procedure. A primary antibody directed against nitrosothiol groups was used in combination with an enzyme-linked secondary antibody. The dye released by the enzyme is insoluble and thus will precipitate close to the site of formation, b S-nitrosylationinaorticrings. Samplesweie treated with Phenylephrine (PE), acetylcholine (Ach), and the inhibitor N-methylarginine (L-NAME) as indicated. Brown stain deposits indicate S-nitrosylation. The traces above the histology panels illustrate the contraction and relaxation responses evoked by the dmgs.

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