Fundamental study detecting apoptosis by the TUNEL (TdT-mediated d-UTP nick end-labeling) method in cytology preparations

We report a fundamental study detecting apoptosis (AP) by the TdT-mediated d-UTP nick end-labeling (TUNEL) method in cytology preparations of a human lymphocyte established cell line (C 5/TK 1), ascites fluid from patients with or without malignancies, and imprint or squash preparations of tissue samples. There is no distinct difference between the conditions using 95% ethanol and 100% methanol as a fixative for routine fixation and AP could be demonstrated even in the cytology preparations which were stored in the fixatives for up to 10 months. On the other hand, prolonged fixation in a formaldehyde fixative (20% formalin) caused nonspecific labeling, and it seemed to be inappropriate for long-time storage. Proteinase K (PK) pretreatment was required for all the fixatives used, and incubation with 10 μg of PK per ml for 15 minutes at room temperature always gave satisfactory results, though incubation with 20 μg of PK per ml or higher concentrations produced cell detachment due to overdigestion. Morphological analysis under light microscopical observation, and comparison of the localization and distribution of AP in cell preparations with those of tissue samples confirmed the reliability of AP detection by the TUNEL method in cell preparations.