Immunohistochemistry fluorescence

Key words Immunohistochemistry, Fluorescence microscopy, IgG, Primary antibody, Secondary antibody, Monoclonal antibody, Polyclonal antibody, Fluorochrome... 

Ge S, Crooks GM, McNamara G, et al. Fluorescent immunohistochemistry and in situ hybridization analysis of mouse pancreas using low-power antigen-retrieval technique. /. Histochem. Cytochem. 2006 54 843-847. 

Ino H. Application of antigen retrieval by heating for double-label fluorescent immunohistochemistry with identical species-derived primary antibodies. J. Histochem. Cytochem. 2004 52 1219-1230. 

Coons AH, Creech H, Jones R (1941) Immunological properties of an antibody containing a fluorescent group. Proc Soc Exp Biol Med 47 200 202 Coons AH, Creech H, Jones R, Berliner E (1942) The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. J Immunol 45 159 170 Dabbs DJ (ed) (2006) Diagnostic immunohistochemistry. Churchill Livingstone, Philadelphia Dairkee SH, Mayall BH, Smith HS, Hackett AJ (1987) Monoclonal marker that predicts early recurrence of breast cancer. Lancet 1 (8531) 514... 

Fluorescence microscopes used in immunohistochemistry are so called epifluorescence microscopes (i.e., excitation of the fluorescence and observation are from above (epi) the specimen). 

Fluorophore absorbs ultraviolet light (or violet, blue or green) and emits light of longer wavelength. Fluorophores are used in immunohistochemistry for labeling primary or secondary antibodies in direct and indirect immunostain-ing methods, respectively. They can be visualized in fluorescence microscopy using special filter sets. 

Fluorescent dyes. Immunofluorescence using fluorescein or rhodamine has been very successfully employed for immunohistochemistry. Fluorescein, when illuminated with UV light, emits a characteristic green fluorescence while rhodamine gives an orange colour. 

Immunohistochemical analyses were performed using antibodies recognizing smooth muscle actin (SMA), troponin-T (Trop-T), troponin T-C (Trop T-C), myosin heavy chain (MHC), muscle actin (MA), a-actinin, a-SR-1, desmin, connexin-43, fibroblasts (all antibodies from Dako, Denmark). For fluorescent immunohistochemistry the AlexaFluor 594 goat anti mouse secondary antibody (Molecular Probes, Leiden, The Netherlands) was used. In addition, cell nuclei were stained using DAPI. 

The avidin-biotin system was developed for detecting antigens at the electron microscope level (Heitzmann and Richards, 1974). Later Heggeness and Ash (1977) proposed the use of this system for fluorescence immunohistochemistry. Guesdon et al. (1979) proposed a variety of labeled avidin-biotin methods which were further supported by Warnke and Levy (1980). The avidin-biotin methods used today are similar to the system described by Hsu et al. (1981). This system is a significant improvement over the previous immuno-histochemical techniques. The problem of endogenous biotin is discussed on page 98. 

Lebeau, A., Deimling, D., Kaltz, C., Sendelhofert, A., Iff, A., Luthardt, B., Untch, M., and Lohrs, U. 2001. HER-2/wen analysis in archival tissue samples of human breast cancer Comparison of immunohistochemistry and fluorescence in situ hybridization.. 1. Clin. Oncol. 79 354-363. 

Pauletti, G., Dandekar, S., Rong, H.-M., Ramos, L., Peng, H., Seshadri, R., and Slamon, D. J. 2000. Assessment of methods for tissue-based detection of the HER-2/wew alteration in human breast cancer A direct comparison of fluorescence in situ hybridization and immunohistochemistry. J. Clin. Oncol. 18 3651-3661. 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

Immunohistochemistry (IHC) is an integral part of both diagnostic and research processes. The history of IHC started in 1941 when bacteria were identified using a direct fluorescent method, and then followed the indirect method, the enzyme-based method with... 

In keeping with current trends in immunohistochemistry to develop alternatives to biotin-streptavidin detection methods, a fluorescyl-tyramide amplification system has recently been introduced (FT-CSA). In this procedure peroxidase is associated with a tissue-bound primary antibody by application of a secondary antimouse Ig antibody to which peroxidase has been conjugated. The peroxidase catalyzes the conversion and deposition of fluorescyl-tyramide onto the tissue section. At this point the reaction can be terminated and viewed by fluorescence microscopy, or the signal can be converted to a colorimetric reaction by the sequential application of an anti-fluorsecein antibody conjugated to peroxidase followed by a diaminobenzidine-hydrogen peroxide substrate. 

Note. ELISA = enzyme-linked immunosorbent assay, FACS = fluorescence-activated cell sorting, HPLC = high-performance liquid chromatography, IHC = immunohistochemistry, LC/MS = liquid chromatography/MS = mass spectrometry, LPS = lipopolysaccharide (endotoxin), NA = not applicable, PAHA = (nonhuman) primate anti-human antibodies. 

Note CHO = Chinese hamster ovary cells, ELISA = enzyme-linked immunosorbent assay, FACS = fluorescent-activated cell sorting or flow cytometry IHC = immunohistochemistry, LBI = ligand-binding inhibition, rec = recovery, RMN = rat micronucleus. 

These assays resemble a hybrid of an immunohistochemistry assay and ELISA. Whole cells are fixed, for example with 3.7% formaldehyde, to MTPs permeabilized by repetitive washing with 0.1% Triton X-100, blocked with a protein solution, probed with primary antibodies (phospho-spe-cific, and non-phospho-specific), washed, and subsequently the secondary antibodies labeled with infrared fluorescent tags are added. After washing, these assays are read in a reader (such as the Odyssey or Aerius) designed for high sensitivity detection of two colors. The two colors are useful because one color can be used to accommodate a stain assigned as a total protein or cell number control or as an antibody to total protein, which allows for normalization. These assays may only require a single antibody versus the dual antibody sandwich required for ELISA. 

The technique of immunohistochemistry is very similar to fluorescence microscopy. This technique differs only in the method of detection or localization of the antibody and can be performed with a conventional light microscope. As with the ELISA and Western blot, the antibody used in this experiment is covalently conjugated to an enzyme, such as horseradish peroxidase. This enzyme is then incubated with a substrate that is converted to an insoluble colored product that will precipitate or deposit at the site of enzyme activity. The distribution and location of the colored product is readily detected with an ordinary light microscope. 

Figure 10.12 Steps involved in immunohistochemistry and a photomicrograph of pancreatic p cells stained for human insulin with a fluorescent label and visualized by fluorescent microscopy.

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