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

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.