Quantitative in situ image analysis of apoptosis in well and poorly differentiated tumors from rat liver

The evaluation of apoptosis is an important aspect in the study of chemical carcinogenesis. Methods were developed employing the ApopTag kit (Oncor, Gaithersburg, MD) and automated image analysis to quantitate the distribution of apoptosis in formalin-fixed, paraffin-embedded liver tumor sections from rats induced by 2-acetylaminofluorene. Specific treatments of tissue sections were developed that permitted quenching of background tissue autofluorescence with crystal violet and permitted permeating the fixed tissue sections by trypsin digestion. Tissue sections were stained by using the ApopTag kit for detection of in situ apoptosis and with propidium iodide as a counterstain for tissue nuclei. Automated statistical evaluation of the percentage of tissue nuclei also staining positively for apoptosis was determined by using dual fluorescence detection and imaging laser microscopy. The quantitative results indicated that the staining index for apoptosis in normal liver was 0.14 +/- 0.04% whereas well and poorly differentiated tumors showed increases of 3.48 +/- 0.59% and 7.41 +/- 0.81%, respectively. The staining indexes for apoptosis showed a tight correlation between fluorescent and peroxidase-diaminobenzidine detection in sequential tissue sections. The use of in situ apoptosis staining and automated image analysis for rapid identification and quantitation of cells undergoing death in fixed tissue will expedite additional studies to evaluate the in situ tissue pathobiology of tumors and aid in the study of molecular mechanisms associated with cancer development and treatment.