Double immunostaining of lymph node sections by monoclonal antibodies using phycoerythrin labeling and haptenated reagents

Double immunostaining of tissue sections by monoclonal antibodies has been performed using, in immunofluorescence, combinations of reagents conjugated with different fluorochromes. These were monoclonal antibodies directly labeled with phycoerythrin (a new red fluorochrome) and haptenated monoclonal antibodies detected by fluorescein-conjugated second or third layers. All the reagents used were commercially available and the double immunostaining was easy to perform and allowed the design of several combinations useful to characterize T-cell subsets in reactive lymph nodes. The results indicate that the simultaneous detection of pairs of antigens by monoclonal antibodies can be applied successfully to tissue sections in order to investigate cellular heterogeneity in normal and pathologic conditions.