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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A073
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.
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