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

A120
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

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.