[Apoptosis--selected methods of detection of apoptosis and associated regulatory factors on tissue sections of tumors].
Methods for identification of apoptotic (AP) cells in tissue sections include light and electron microscopy and immunohistochemical (IHC) detection of apoptotic antigens. Bcl-2 at many tumors inversely correlates with AP and is an indirect marker of AP index. Transglutaminase is often expressed in nonapoptotic cells, and thus represents a non specific marker of AP. Likewise, expression of FAS does not necessarily represent a transformation into AP. IHC detection of caspases does not distinguish between active and nonactive forms of the proteases. Immunolabeling of biotin-conjugated Annexin V is used for the identification of phosphatidylserine residues exposed on the surface of AP cells. Annexin V immuno-gold labeling by means of electron microscopy will allow a more refined description of the morphological events occurring during apoptosis. TUNEL, ISEL and ISNTA methods detect DNA breaks. The rate of AP detected by TUNEL is about 20% higher then by apoptotic figure counting. DNA strand breaks can also occur during DNA repair, electrocoagulation, autolysis, fixation and paraffin embedding. With Apostain, DNA is selectively denaturated by heating with formamide and stained by monoclonal antibody specific to single-strand DNA. It specifically stains condensed chromatin of apoptotic cells. M30 IHC uses a monoclonal antibody binding to the product resulting from cleavage of cytokeratin 18 by activated caspases. M30 is negative in necrotic cells and in progressively degraded cells (AP bodies). In contrast to some pilot studies, we have not reached sufficient sensitivity and specificity of IHC detection with M30 (Roche) in breast carcinomas.