General suitability of techniques for in situ detection of apoptosis in small intestinal epithelium

The present study was designed to evaluate different techniques for the in situ detection of apoptosis in human and rat small intestinal epithelium. The techniques included light microscopy (LM) and transmission electron microscopy (TEM) observation of epoxy resin-embedded tissue, scanning electron microscopy (SEM), TUNEL assay, and antibodies directed against caspase cleavage products of caspase 3, cytokeratin 18 (CK 18), and apoptotic single-strand DNA (ssDNA). All techniques, if the labeling was positive, showed apoptotic cells exclusively at the villus tip. LM and TEM were the most reliable and revealed morphological signs typical of cells that have died via apoptosis. SEM indicated the extension of the process. The antibody recognizing cleaved caspase 3 could be considered an appropriate marker for apoptotic epithelial cells in human and rat small intestine. However, the majority of epithelial cells lining the proximal small intestinal villus contained only low levels of intact CK 18. Therefore, sufficient amounts of cleaved CK 18 for immunohistochemical detection were not generated during apoptosis, rendering the application of the antibody inappropriate. The antibody detecting formamide-denatured ssDNA in apoptotic cells was both suitable and reliable; however, the particular staining procedure used compromised the tissue preservation. In comparison to this, the TUNEL assay was less reliable. Although it was performed with a commercially available ready-to-use kit, its application conditions had to be adjusted for each specimen on the basis of the findings produced by other techniques.