COMBINED IMMUNOCYTOCHEMISTRY AND FLUORESCENCE IN-SITU HYBRIDIZATION FOR SIMULTANEOUS TRICOLOR DETECTION OF CELL-CYCLE, GENOMIC, AND PHENOTYPIC PARAMETERS OF TUMOR-CELLS

We describe the development and application of a sensitive high-resolu tion fluorescence alkaline phosphatase (APase)-Fast Red immunocytochem ical (ICC) staining method in combination with fluorescence in situ hy bridization (ISH) and bromodeoxyuridine (BrdU) detection. The high flu orescence intensity, accurate localization, and advantageous slow-fadi ng properties make the APase-Fast Red reaction a valuable tool for det ection of antigens or specific DNA probes in biological cell preparati ons. Since the enzyme precipitate proved to be resistant to enzymatic pre-treatment steps and stable during the entire ISH procedure, APase- Fast Red immunostaining could be combined with subsequent visualizatio n of DNA target sequences by fluorescence ISH. The lung cancer cell li nes NCI-H82 and EPLC 65 were used as a model system for simultaneous d etection of cell proteins, such as the neural cell adhesion molecule ( N-CAM), cgtokeratin filaments, lamin or the Ki67 antigen (Ki67;Ag), an d centromere-specific DNA probes for human chromosomes 1, 7, or 17. In addition, the combined ICC/ISH procedure could be extended with the i mmunodetection of BrdU incorporated by tumor cells in S-phase. As a co nsequence, a combined ICC/ISH/BrdU detection procedure is now availabl e that enables analysis of relatively complex tumor populations on the basis of different ICC and genetic markers as well as proliferative a ctivity.