6th ESACP Congress, Heidelberg, April 7-11, 1999

A073
COMPARISON OF DIFFERENT BICOLOR ISH DETECTION METHODS TO ANALYZE CHROMOSOMAL ABERRATIONS IN INTERPHASE CELLS
Hopman AHN, Kamps M, Haesevoets A, Speel EJ, Ramaekers FCS

Dept.Molecular Cell Biology & Genetics, Dept. Internal Medicine, Academic Hospital Maastricht, University Maastricht, Netherlands, Dept. Pathology, Univ.Hospital Zürich, Switzerland

We used biotin and digoxigenin labeled repeat sequence probes to compare different bicolor detection methods with respect to reproducibility and sensitivity. As a model the cell line T24 and formalin fixed tissue sections were used. In the latter case the sensitivity was most critical. The detection methods included: 1) fluorescence detection and 2) bright field microscopic detection based on indirect immunocytochemical procedures ranging from 4 to six immunolayers. The latter detection includes a peroxidase-diaminobenzidine (PO-DAB, brown) and a peroxidase-tetramethylbenzidine/tungstate (PO-TMB) staining. 3) the catalyzed reported deposition (CARD) amplification system comprising only 2 incubation steps. The double-target ISH with the CARD detection was based on peroxidase (PO) labeled avidin and sheep anti-digoxigenin. The cytochemical detection was followed by CARD amplification with rhodamine and fluorescein labeled tyramides. Between the PO incubation steps an inactivation step was performed to block the residual peroxidase activity of the first layer prior to the second peroxidase reaction. Compared with the indirect immunocytochemical detection procedure the processing time was reduced with 70%, the signal intensity was strongly enhanced and evaluation using a low microscopical magnification was strongly improved.