Simultaneous detection of DNA fragmentation (apoptosis), cell proliferation (MIB-1), and phenotype markers in routinely processed tissue sections

In situ DNA fragmentation assays have proved to be particularly useful in t he detection of apoptosis in routinely processed, paraffin-embedded tissue sections. In the present study, a triple-antigen labelling technique was pe rformed to demonstrate DNA fragmentation (apoptosis), cell proliferation (M IB-1), and phenotypic markers in the same tissue section. The in situ apopt osis assay was conducted with the TUNEL method developed by a avidin-biotin alkaline phosphatase complex (ABcomplex/AP). The proliferation-associated MIB-1 antigen was demonstrated in the second staining sequence by the avidi n-biotin peroxidase method (ABC). The phenotypic markers chromogranin A or prostate-specific antigen (PSA) were visualized by the alkaline phosphatase anti-alkaline phosphatase method (APAAP) in the third staining sequence. T he feasibility of this triple-labelling technique was tested in formalin-fi xed, paraffin-embedded tissue of prostatic adenocarcinomas from 8 patients with recurrent, hormone-refractory disease. Although these tumours revealed marked neuroendocrine differentiation, cell proliferation and apoptosis we re detected exclusively in non-endocrine (chromogranin A-negative) tumour c ells that expressed PSA variably. The triple-labelling protocol described h ere allows the phenotypic characterization of proliferating and apoptotic c ell populations in the same tissue section. It may be useful in studies of tissue kinetics in physiological and pathological processes.