title: A novel triple-color detection procedure for brightfield microscopy, combining in situ hybridization with immunocytochemistry.
abstract: We describe a fast light microscopic procedure for the simultaneous enzyme cytochemical detection of three different DNA target sequences in contrasting colors in both interphase and metaphase cell preparations. Chromosome-specific DNA probes labeled with either biotin, digoxygenin, or fluorescein were hybridized as a mixture and detected clearly and accurately by precipitates of the horseradish peroxidase-diaminobenzidine (PO-DAB, brown color), alkaline phosphatase-Fast Red (APase-Fast Red, red color), or horseradish peroxidase-tetramethylbenzidine (PO-TMB, green color) reaction, respectively. The PO-TMB reaction product was stabilized effectively by the addition of sodium tungstate to the reaction mixture, thus making the PO-TMB reaction now generally applicable to in situ hybridization (ISH). To avoid mixing of the precipitates of the two PO reactions used in the triple-color ISH method, the first detected PO activity was always completely inactivated by a mild acid treatment before the second one was applied. Finally, the cell preparations were embedded in a thin protein layer cross-linked by formaldehyde to ensure permanent stabilization of the enzyme reaction products and optimal visualization of color contrast. The triple-color ISH detection procedure could be combined with beta-galactosidase-5-bromo-4-chloro-3-indolyl-beta- D-galactoside (beta-Gal-BCIG) immunocytochemistry (ICC), leading to the simultaneous localization of multiple DNA targets and a protein target in the same cell. The described procedure may therefore be a valuable tool in the areas of cytogenetics, cell biology, and molecular pathology.
authors.affiliation: Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht, The Netherlands.
authors.forename: E J
authors.initials: EJ
authors.lastname: Speel
authors.forename: M P
authors.initials: MP
authors.lastname: Jansen
authors.forename: F C
authors.initials: FC
authors.lastname: Ramaekers
authors.forename: A H
authors.initials: AH
authors.lastname: Hopman
mesh_terms: D002478:Cells, Cultured; D006801:Humans; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization / Q000379:methods*; D008853:Microscopy / Q000379:methods*; D014407:Tumor Cells, Cultured