Cellular antigens detection

The specificity of antibodies can be exploited in order to probe the in situ organization of cells and tissues. Cellular antigens can be identified both in viable cells and in frozen or fixed tissue sections. Antibodies are used to identify the appropriate antigen in the section and then the position of this primary antibody may itself be detected either directly if it was initially labelled or indirectly using another secondary antibody or molecule to attach to the antibody (Figure 7.8). Samples need to be carefully washed after addition of the primary or labelled antibody in order to prevent any non-specific reactions. Labels that have been successfully linked to antibodies include the following ... 

Described here is an indirect method for detecting two different cellular antigens in acetone-fixed tissue, using a rabbit polyclonal antibody, and a murine monoclonal antibody on the same section. One secondary antispecies antibody is conjugated with alkaline phosphatase, the other with peroxidase, thus resulting in two differently colored products showing the localization of the two antigens (Fig. 1). 

In 1942 Coons et al. described an immunofluorescence technique for detecting cellular antigens in tissue sections [15].This method utilized a fluorescent label bound to the primary antibody to localize the target antigen. In this case the fluorescent label was the detection method. The use of an immunoenzyme approach to detect binding of the primary antibody was introduced a quarter of a century later [16] with the introduction of peroxidase-labeled antibodies. 

The following part provides an overview of the most common antibodies used for tumor diagnosis as well as the expression pattern of targeted antigens detected by the immunohistochemical reaction. The expression pattern of the different cellular antigens is an important factor to consider and essential for the precise interpretation of the immunohistochemical stains and includes the following expression (stain) patterns ... 

Proteins are usually detected by immunohistochemical procedures. Some proteins can be detected directly (e.g. by their errzymatic activity). However, the number of detected proteins depends on the availability of specific antibodies and often determined by the quality and the quantity of the surgical specimen. Immunohistochemical procedures discussed in the prior section have the advantage that the morphology of the tumor is still conserved and can be studied simultaneously with additional information regarding cellular antigens and their localization. In addition to immunohistochemistry, modern proteomics offers new approaches to detect proteins by western blotting, 2-dimensional gel electrophoresis or by protein arrays. 

In Protocol IB, a soluble whole cell extract is produced by lysis in RIPA, a mixed micelle detergent-containing buffer. RIPA buffer results in extraction of proteins without complete denaturation and cellular antigens are maintained in conformations that can be detected by immrmopredpitation (Protocol 14B). How-... 

Figure 1 The steps involvea in detection of cellular antigen by immunoblotting. Individual steps are described in the text.

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