Fluorescence ratio measurements of double-labeled probes for multiple in situ hybridization by digital imaging microscopy.

摘要: To expand the multiplicity of in situ hybridization (ISH) procedure, which is presently limited by number fluorochromes spectrally separable microscope, a digital fluorescence ratio method proposed. For this purpose, chromosome-specific repetitive probes were double-labeled with two haptens and hybridized to interphase nuclei human peripheral blood lymphocytes. The immunocytochemically detected specific antibodies conjugated FITC or TRITC. TRITC intensities spots obtained different double-haptenized measured, was calculated for each ISH spot. Combinations haptens, such as biotin, digoxigenin, fluorescein, sulfonate, acetyl amino fluorene (AAF), mercury (Hg) used. intensity (FITC/TRITC) fairly constant all combinations used, coefficients variation between 10 30%. study feasibility probe identification procedure on basis hapten ratios, one varying relative concentrations modified nucleotides (biotin-11-dUTP digoxigenin-11-dUTP) nick-translation reaction. Measurement showed that concentration used labeling procedures reflected mean spots. Furthermore, distributions little overlap due relatively small variation. results indicate multiple based imaging feasible.