Immunological staining

Sensitivity of immunological stain = 96% = 0.96 False-negative error rate of the test = 4% = 0.04 Specificity of the test = 94% = 0.94 False-positive error rate of the test = 6% = 0.06 Prevalence of effect in the tissues = 1% = 0.01... 

FIGURE 28-36 Distribution of a maternal gene product in a Drosophila egg. (a) Micrograph of an immunologically stained egg, showing distribution of the bicoid (bed) gene product. The graph measures stain intensity. This distribution is essential for normal develop-... 

Immunological staining of primary spermatocyte nuclei Protocol 43... 

Duan, L., Wang, Y., Li, S.S-C., Wan, Z., and Zhai, J. (2005) Rapid and simultaneous detection of human hepatitis B virus and hepatitis C virus antibodies based on a protein chip assay using nano-gold immunological amplification and silver staining method. BMC Infect. Dis. 5, 53. http //www.biomedcentral. com/1471-2334/5/53. 

Endometriosis is an estrogen-dependent disorder mostly occurring in reproductive-age women characterized by a growth of the endometrium outside the uterine cavity (Oral et al. 1997 Child et al. 2001). Explanations of how the tissue stains this abnormal placement are controversial, although the predominant theory is that retrograde menstruation is the cause (Oral et al. 1997 Child et al. 2001). Additional factors that maybe pivotal in the disease s pathogenesis include immunologic abnormalities, endometrial disorders, and peritoneal dysfunction (Oral et al. 1997 Child et al. 2001). 

A program employing a immunochemical stain based test to screen tissues for a specific effect will be discussed as an example. This test uses small amounts of antibody tissues for a specific effect, and the presence of an immunologically bound stain is considered a positive result. If the sensitivity and specificity of the test and the prevalence of biochemical effect are known, Bayes theorem can be used to predict what proportion of the tissues with positive test results will have true-positive results (actually be effected). 

A collection of useful protocols for the simultaneous immunoenzyme staining of two or more tissue antigens is available on the websites http //www.protocol-online.org/prot/Immunology/ http //www.vectorlabs.com/Protocols/MLB.pdf and http //www.ihcworld.com/. The protocols given in this chapter are practiced in the authors laboratory. These methods fall into two main categories (a) simultaneous immunoenzymatic double staining, and (b) sequential immunoenzymatic double/ multiple staining. 

The problem has now been solved, and it is possible to measure the phase angle of the probe as the cells pass through the laser beam.09,40) While these measurements have not yet been applied to Ca2+, the method has been validated with standard beads and stained cells. In our opinion, this new flow cytometry parameter, and our increasing knowledge of lifetimes of probes, will result in the increased use of flow cytometry for studies of intracellular physiology, in addition to the current emphasis in immunology, cell activation, and ploidy. 

Fig. 1. Photomicrographs of a reactive lymph node follicle with germinal center (G), mantle (M), and surrounding paracortex (P) immunostained using the avi-din-biotin complex technique. (Top) Follicle stained with an antibody specific to B-cells, B1 (CD20) (Coulter Immunology, Hialeah, FL), counterstained with Methyl Green. (Bottom) Parallel section of the same follicle stained with an antibody specific to T-cells, Leu 4 (CDS) (Becton-Dickinson, Mountain View, CA). Scale bar = 100 p.

It was shown by Creech and Jones (1) in 1940 that proteins, including antibodies, could be labeled with a fluorescent dye (phenylisocyanate) without biological or immunological effects to the intended target. In theory, fluorescent reporters (tracers, probes, antibodies, stains, and so on) can be used to detect or measure any cell constituent, provided that the tag reacts specifically and stoichiometrically with the cellular constituent in question (2). Today, the repertoire of fluorescent probes is expanding almost daily see Chapter 14). One area that has benefited from the ever-increasing number of fluorescent probes is flow cytometry. 

Hosoda H, Ishikawa E (1993) Coupling of haptens to carriers and to labels. In Masseyeff RF, Albert WH, Staine NA (eds.) Methods of immunological analysis. Samples and reagents. VCH, Weinheim, p... 

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