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.
Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands.
E J
EJ
Speel
J
J
Herbergs
F C
FC
Ramaekers
A H
AH
Hopman
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