In situ end labelling: effect of proteolytic enzyme pretreatment and hydrochloric acid.

Abstract

AIM: To determine the effect of dilute hydrochloric acid on the in situ end labelling (ISEL) reaction, with and without a variety of different proteolytic enzymes. METHODS: Sections of tissue fixed in buffered formalin were pretreated with trypsin, protease XIV (at two different concentrations), or protease XXIV (for two different incubation times), with and without subsequent 1 M hydrochloric acid treatment. The results were compared with those obtained using hydrochloric acid alone, with proteinase K, and pepsin pretreatment, and with no pretreatment. RESULTS: When hydrochloric acid was added to the sections in addition to trypsin, protease XIV, and protease XXIV, there was a significant increase in ISEL reactivity in both apoptotic nuclei and morphologically normal nuclei. Hydrochloric acid alone had no significant effect. CONCLUSIONS: Hydrochloric acid has a distinctive effect on the ISEL reaction that is dependent on prior proteolytic digestion.