Quantitative assessment of cardiac myocyte apoptosis in tissue sections using the fluorescence-based tunel technique enhanced with counterstains

Apoptosis is a distinct form of cell death, induced, for example, by ischae mia/reperfusion injury, that results in characteristic alterations in cell morphology and fate. In tissue sections, the most commonly used technique t o detect apoptosis is terminal deoxynucleotidyl transferase mediated nick e nd labelling (TUNEL) staining which labels the ends of DNA strand breaks ch aracteristic of the apoptotic process. However, without the employment of a dditional staining, TUNEL is only a qualitative procedure that gives no inf ormation about the proportion of negative cells nor the cell type undergoin g apoptosis. We have utilised propidium iodide (PI) as a counterstain to vi sualise TUNEL negative nuclei together with anti-desmin antibody in order t o assess quantitatively apoptosis in specific cell types. The procedure has been evaluated in tissue sections from isolated perfused rat hearts subjec ted to ischaemia and reperfusion. Hearts were cross-sectioned into four 2.5 mm thick slices which were fixed in 4% formaldehyde and embedded in paraff in. Serial sections (5 microns) were cut, dewaxed and pretreated by incubat ion with trypsin at 37 degrees C for 30 min. After the employment of the TU NEL assay, sections were labelled with anti-desmin antibody, counterstained with PI and finally examined by confocal fluorescent microscopy. Apoptosis was not seen in sections from hearts subjected to ischaemia alone nor in c ontrol hearts. After 35 min of ischaemia the percentages of TUNEL positive cells were very low both in myocytes (0.1%) and in non-myocytes (0.3%). In ischaemic-reperfused hearts, the number of TUNEL positive cells was only si gnificantly higher in vascular cells (44 +/- 5%) and cardiac myocytes (6 +/ - 2%). This simple method therefore allows quantification of apoptosis in m yocytic and non-myocytic cells in tissue sections. Use of alternative immun ohistochemical markers would permit adaptation of the method to the quantit ative assessment of apoptosis in other tissues. (C) 1999 Elsevier Science B .V. All rights reserved.