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TUNEL Apoptotic Cell Detection in Tissue Sections: Critical Evaluation and Improvement
Françoise Labat-Moleura, Christiane Guillermeta, Philippe Lorimiera,b, Catherine Roberta,b, Sylvie Lantuejoula, Elisabeth Brambillaa,b, and Adrien Negoescuba Laboratoire de Pathologie Cellulaire, CHRU, Grenoble, France
b Groupe de Recherche sur le Cancer du Poumon, Institut Albert Bonniot, Faculté de Médecine, Grenoble, France
Correspondence to: Adrien Negoescu, Groupe de Recherche sur le Cancer du Poumon, Institut Albert Bonniot, Faculté de Médecine, Domaine de la Merci, 38706 Grenoble cedex, France.
TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 70–80% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, step-by-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens. (J Histochem Cytochem 46:327–334, 1998)
Key Words: TUNEL, sensitivity, specificity, microwaves, pH, preapoptosis
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