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6th ESACP Congress, Heidelberg, April 7-11, 1999 |
A120
We developed a catalyzed reporter deposition (CARD) procedure for the
simultaneous detection of the anti-apoptosis antigen bcl-2 and the cell
proliferation antigen Ki-67 in immunohistochemistry assays by means of
differently fluorochrome-labeled tyramides. We have sequentially applied
differently labeled (AMCA, FITC, TRITC) fluorochrome-conjugated tyramides
to improve the detection of the different antigens in frozen sections of
normal cervical lesions. A ten-to 1.000-fold increase in staining efficiency
was achieved, depending on the antibody, by visualizing the in situ localized
peroxidase activity, initiated by the deposition of fluorochrome-labeled
tyramide molecules. To allow specific deposition of the second tyramide
conjugate for bicolor immunofluorescence assays, complete inactivation of
the remaining peroxidase was obtained by mild acid treatment followed by a
formaldehyde fixation step. High specific and intense fluorescent signals
were obtained for both antigens. The fluorescence CARD detection system
provided a considerable increase in sensitivity compared to conventional
bicolor immunofluorescence assays. The method is quick and allows an
efficient amplification tool for multicolor immunofluorescence staining.
MULITCOLOR FLUORESCENT LABELING IN IMMUNOCYTOCHEMISTRY USING CATALYZED
REPORTER DEPOSITION (CARD) AMPLIFICATION
Schaart G, Ramaekers FCS, Hopman AHN
Dept. Molecular Cell Biology & Genetics, University Maastricht, Netherlands