Improved detection of apoptotic cells using in situ TUNEL technique

CUELLO CARRIÓN F. D.; CIOCCA D. R.

Philadelphia, PA, USA

Congreso; 90th Annual Meeting of the American Association for Cancer Research; 1999

American Association for Cancer Research

We are studying the effect of neoadyuvant chemotherapy in biopsy samples from cervical and breast carcinoma patients. Apoptosis is detected in formalin fixed, paraffin sections, by morphological characteristics (on hematoxylin and eosin stained sections) and applying the Apop Tag ™ Plus detection kit (Oncor, Gaithersburg, MD.). As positive control we utilize prostate glands from castrated rats. Using the standard protocol supplied with the kit, the number of apoptotic cells by the in situ TUNEL technique was inferior to the number of cells detected on H&E-stained sections. Microwave irradiation of tissue sections showed little improvement. In contrast, a more sensitive procedure was obtained when the antibody provided with the kit was replaced by a mouse monoclonal anti-digoxin antibody conjugated with biotin (Sigma Chemical Company, St. Louis, MO.). Thereafter, the samples were incubated with a secondary antibody, biotinylated rabbit anti-mouse IgG (whole molecule) (Dako Corporation, Carpinteria, CA.), and the signal was amplified using peroxidase-Iabeled streptavidin (LAB Complex) (Dako). With these modilications in the technique, in the rat tissues as well as in the human cancer samples, a considerable improvement in the detection of apoptotic cells was observed.