Tissue-bound antibody

Wash cultured cells attached to 35-mm plastic tissue-culture dishes in Dulbecco s PBS, then incubate in a blocking buffer consisting of BSA-PBS for 5 min, and cool to 4°C (see protocol flow chart in Fig. 1). Cooling prevents subsequent endocytosis of any added antibody reagents, as well as minimizing lateral mobility of bound antibody in the plane of the plasma membrane (see Notes 5 and 6). 

Proteins can be detected in tissue sections or cell cultures using similar immune detection systems. Use of an antibody to detect specific proteins in tissues is called immunohistochemistry, whereas detection of proteins in cell suspensions is called immunocyto-chemistry. Tissues can be prepared by fixation and embedding in paraffin wax, or by rapid freezing in a compound that inhibits ice formation in the tissue, so as to preserve cell morphology. Some antibodies do not work well with paraffin-embedded tissues, probably because the antibody cannot access the antigen properly (133). The most common labeling system used for detection of the bound antibody is an enzyme-coupled secondary antibody that produces a color reaction... 

Immunohistochemical staining of tonsillitis, gastric adenocarcinomas, and breast carcinomas can be obtained using MIB-1 antibody in conjunction with EDTA-NAOH solution and a pressure cooker (Kim et ah, 1999b). EDTA solution is thought to be more effective than other buffers in unmasking the epitopes, presumably because it removes (chelates) tissue-bound calcium ions. 

Streptavidin (from Streptomyces avidinii) and avidin (from chicken egg) both possess four binding sites for biotin. The biotin molecule is easily conjugated to antibodies and enzymes. In the avidin-biotin complex (ABC) method secondary antibodies are conjugated to biotin and function as links between tissue-bound primary antibodies and an avidin-biotin-peroxidase complex (5). 

EPOS, which contained primary antibodies, the EnVision system contained secondary antibodies with anti-mouse Ig and anti-rabbit Ig specificity. This universal reagent could be used to detect any tissue-bound primary antibody of mouse or rabbit origin. The utility of this method opened the door to a new family of polymer-based immunohistochemical methods. The sensitivity of these methods compared to LSAB and ABC methods was comparable or even slightly greater in most cases (7). With the latest development of EnVision FLEX+ the sensitivity has been improved even further. However, because of the large molecular size of the polymer conjugates, accessibility to certain epitopes was restricted, presumably due to steric hindrance, in a minority of cases. 

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. 

An immunogen is either a protein or a substance coupled to a carrier, usually a protein, that when introduced into a foreign host is capable of inducing the formation of an antibody in the host. The route of introduction of the immunogen is usually, but not always, intradermal. The antibody produced may be either circulating (humoral) or tissue bound (cellular), as in delayed hypersensitivity reactions or graft host reactions. 

Adequate binding affinity for tumor localization has not been defined, although the importance of high affinity for maximal cell binding and for sensitive in vitro radioimmunoassay has been shown (41-43). It has, however, also been suggested that low affinity is preferable in vivo to allow more tissue penetration and avoid trapping of tightly bound antibody at the tumor surface... 

In this method, samples of freshly excised tissues were fixed by boiling them in a 10% neutral formaldehyde solution for 5 min. The fixed tissues were then embedded in paraffin and cut into 3 pm thick sections. The sections on glass slides were deparaffinized and dehydrated by passage through xylene and a graded alcohol series. Then they were incubated with a primary antibody against poly(ADP-ribose). The antibody used, IgG monoclonal antibody lOH, recognized the linear structure of poly(ADP-ribose) described previously (3). Bound antibody was stained with die avidin biotin peroxidase complex or by an indirect immunofluorescence technique. 

Although several types of fluorescent beads were proposed as a microscopic fluorescence standard 30 years ago,2 beads have not been used as a proteinembedding matrix for routine IHC on FFPE tissue. We recently tested primary coated beads ( Dynabeads, Dynal, New York) that are coated with a goat anti-mouse antibody on the surface of the beads. In the first experiment, a monoclonal antibody to cytokeratin 7 (DAKO, 50pL/34.5pg) was bound to the beads by incubating with the beads (150 pL at a concentration of 109 beads/1 pL) at 4°C in a cold room with an automatic shaker for overnight. Incubation was followed by three phosphate-buffered saline (PBS) washes,... 

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