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-resolution fluorescence alkaline phosphatase (APase)-Fast Red immunocytochemical (ICC) staining method in combination with fluorescence in situ hybridization (ISH) and bromodeoxyuridine (BrdU) detection. The high fluorescence intensity, accurate localization, and advantageous slow-fading properties make the APase-Fast Red reaction a valuable tool for detection of antigens or specific DNA probes in biological cell preparations. 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 visualization of DNA target sequences by fluorescence ISH. The lung cancer cell lines NCI-H82 and EPLC 65 were used as a model system for simultaneous detection of cell proteins, such as the neural cell adhesion molecule (N-CAM), cytokeratin filaments, lamin or the Ki67 antigen (Ki67-Ag), and centromere-specific DNA probes for human chromosomes 1, 7, or 17. In addition, the combined ICC/ISH procedure could be extended with the immunodetection of BrdU incorporated by tumor cells in S-phase. As a consequence, a combined ICC/ISH/BrdU detection procedure is now available that enables analysis of relatively complex tumor populations on the basis of different ICC and genetic markers as well as proliferative activity.
Speel
E J
EJ
Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands.
Herbergs
J
J
Ramaekers
F C
FC
Hopman
A H
AH
D000469:Alkaline Phosphatase; D001973:Bromodeoxyuridine; D002453:Cell Cycle; D015342:DNA Probes; D004273:DNA, Neoplasm / Q000032:analysis; D006801:Humans; D007150:Immunohistochemistry; D017404:In Situ Hybridization, Fluorescence; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009363:Neoplasm Proteins / Q000032:analysis; D009369:Neoplasms / Q000235:genetics / Q000473:pathology*; D009499:Neutral Red; D010641:Phenotype; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D015342:DNA Probes; D004273:DNA, Neoplasm; D009363:Neoplasm Proteins; D009499:Neutral Red; D000469:Alkaline Phosphatase; D001973:Bromodeoxyuridine