Specificity of TUNEL method depends on duration of fixation

Recent evidence has suggested that apoptosis plays an important role in various diseases, but concerns about the specificity of the TUNEL method for detecting apoptosis have been raised. The purpose of the present study was to investigate the specificity of the TUNEL method by using immersion and perfusion fixed tissues from both normal rats and rats with heart failure. Although a few positive cells were observed in perfusion fixed tissues, a significant number of positive cells were observed in immersion fixed tissues, especially when fixed tissues were kept for an extended time before the TUNEL assay was applied. The results of TUNEL staining should be interpreted with caution. When immersion fixation is used, fixed tissues should be assayed by the TUNEL method before the DNA degenerates.