Double immunofluorescence labelling of routinely processed paraffin sections

Double immunoenzymatic labelling of routinely processed human tissues has b een used in many histopathology laboratories to compare the expression patt erns of pairs of antigenic markers. However, these techniques are time-cons uming, prone to background staining, and rarely suitable for detecting two antigens present at the same site, since one label tends to obscure the oth er. This paper reports the use of immmunofluorescence for double labelling of pairs of molecular markers in routinely processed tissue. The primary an tibodies are either monoclonal reagents of differing isotype/subclass, or a ntibodies from different species, and labelling is visualized on a conventi onal fluorescence microscope equipped with a cooled CCD camera. Images can be captured and adjusted using personal computer hardware and software. Thi s approach could be used for a wide range of tissue markers and only minima l tissue autofluorescence was observed. The procedure is more rapid than en zyme-based techniques and avoids the problems of interpreting two antigens present at the same site. Its establishment involves relatively minor expen diture and it may represent the optimal technical approach to the co-locali zation of pairs of antigens in routinely processed tissue samples. Copyrigh t (C) 2000 John Wiley & Sons, Ltd.