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 the detection of apoptosis in routinely processed, paraffin-embedded tissue sections. In the present study, a triple-antigen labelling technique was performed to demonstrate DNA fragmentation (apoptosis), cell proliferation (MIB-1), and phenotypic markers in the same tissue section. The in situ apoptosis 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 avidin-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. The feasibility of this triple-labelling technique was tested in formalin-fixed, 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 were detected exclusively in non-endocrine (chromogranin A-negative) tumour cells that expressed PSA variably. The triple-labelling protocol described here allows the phenotypic characterization of proliferating and apoptotic cell populations in the same tissue section. It may be useful in studies of tissue kinetics in physiological and pathological processes.