Postmortem delay and temperature conditions affect the in situ end-labeling (ISEL) assay in brain tissue of mice

Apoptotic cell changes occurring under certain developmental, physiological, and pathological conditions have been of increasing interest during recent years. Due to occasional difficulties in detecting apoptosis in routinely stained sections, various methods have been developed to facilitate tissue examination. Fragmentation of DNA during the process of apoptosis is a prerequisite for detection in the in situ end-labeling (ISEL) procedure. It is yet unclear whether other mechanisms of cell change that induce DNA fragmentation such as necrosis and postmortem autolysis also show positive staining with the ISEL technique. To investigate whether the ISEL assay visualizes autolytic DNA changes along with apoptotic DNA fragmentation, we tested the technique on brain tissue of mice after different time intervals (0, 6, 12, 24, 48, 72 h) of postmortem delay (PMD) and at 2 different temperatures of postmortem storage (4 degrees C and room temperature (RT)). Our semiquantitative results show that up to 24 h of PMD no prominent difference in labeling is observable at both temperatures. After 48 and 72 h of PMD at RT clusters of labeled cells begin to appear. Clusters of stained cells should therefore not be considered as apoptosis when using the ISEL assay.