Introduction 1972 1972 1980 2007 1 Fig. 1 Transmission electron microscopic image of an apoptotic cell in a human kidney biopsy. Note the pyknotic, shrunken nucleus and the very condensed cytoplasm  In more recent years, evaluation of cells and tissues for apoptosis has evolved towards staining for light microscopic and flow cytometric analysis. As the biochemical and cell signaling events involved in the apoptotic cascade have been revealed, new tools for the analysis of apoptosis have emerged. Many of these tools are in the form of antibodies raised against proteins specific for the apoptotic pathway, or against neoepitopes on proteins resulting from action of an activated enzyme. While these new probes may specifically target aspects of the apoptotic pathway, they do not address the ultrastructural changes upon which the term apoptosis was originally defined. 1999 2001 2002 Classification of cell death 2007 2007 Pathways of apoptosis 1998 c asp ases F a d d 2003 1998 c  2000 c  2000 2001 1996 2005 2007 2002 2003 Detection of apoptosis by electron microscopy 1972 1980 2007 1980 2007 Detection of apoptosis by cytochemical optical microscopy 1 Table 1 Commercial sources for apoptosis reagents Reagent Commercial source TUNEL assay kit     Apo stain (F7-26) (anti-ssDNA) Axxora, LLC, San Diego, CA Anti-cleaved caspase 3 (ASP 175) Cell Signaling Technology, Inc., Danvers, MA Anti-cleaved cytokeratin 18 Chemicon, Temecula, CA Annexin V-FITC   d d    c   Anti-apoptosis-inducing factor  This list is not meant to be exhaustive, but to serve as a starting point to search for reagents. Apologies to those companies not listed The TUNEL assay 1999 2000 2001 2002 2003 2006 2006 1998 1999 1999 1996 1998 1998 2000 1996 1998 The TUNEL assay maintains its popularity as a marker of apoptotic cells; it is available in a kit form, is easy to use, and has been available for a number of years, becoming entrenched in the literature. However, given the concerns raised above with its specificity for apoptotic cells, we are of the opinion that other methods should be employed concomitantly with this assay in order to make a determination that the mode of cell death observed is the result of an apoptotic mechanism. Anti-single-stranded DNA antibody (Apostain) 1994 1996 2001 2001 2006 2001 2003 2004 Apoptosis-related neoepitopes 2001 2002 2007 2 3 2007 1980 Fig. 2 green red arrowheads solid arrows blue  Fig. 3 red blue  Annexin V 1994 1999 Other probes for apoptosis 2004 2005 2003 d d c  c  1999 c  c  c  1998 2000 2000 2000 2000 2004 Detection of apoptosis by video optical microscopy 1997 1999 1997 1997 2002 2002 Detection of apoptosis by laser scanning cytometry 1991 1997 2001 2006 1999 6 2001 4 4 Fig. 4 Upper panel  green  orange red  orange red  orange red  Middle panel  green  orange red  orange red  orange red  2 2 2 2 Bottom panel  middle panel 2001  Detection of apoptosis by atomic force microscopy (AFM) 1986 x y 2006 1994 1997 2003 2006 2005 An example of the importance of delineating apoptosis: cell death in the heart 1972 1974 1980 1972 2007 2006 2007 1998 In view of these considerations, it appears important to determine definitively whether the presence of ssDNA coupled with the presence of caspace 3 cleavage product does, indeed, map isomorphically with the cells that exhibit electron microscopic criteria of apoptosis. Furthermore, it will be important to determine the extent to which the positivity of each of these criteria of apoptosis occurs in hearts of experimental animals subjected to ischemia for selected intervals followed by reperfusion for selected intervals as well as in those from animals subjected to myocardial ischemia that is persistent and to define the time course of the evolution of apoptosis, if present, under both conditions. It is, of course, possible that apparently scanty apoptosis at any given time could be of considerable biological importance since apoptotic cells are rapidly destroyed or removed by macrophages. Thus, if a given percentage of cells, though a small one, were exhibiting apoptosis repetitively, the cumulative amount could be substantial. Accordingly, both the temporal and the spatial evolutions of apoptosis associated with myocardial ischemia and infarction require elucidation. This is particularly important because apoptosis in the heart may be secondary to necrosis rather than a primary mode of cell death, a distinction that needs to be clarified because of the disparate therapeutic implications of the two possibilities. Only when armed with information needed to characterize apoptosis in the heart more definitively will it be possible to delineate the potentially beneficial effects of tissue protective interventions designed to retard or prevent apoptotic cell death in cardiomyocytes subjected to ischemia. Concluding remarks 2006 Accordingly, the take-home message for any morphological or cytochemical investigation of apoptosis is to employ more than one detection method and run multiple controls.