A rapid staining procedure for two-color analysis of lymphocyte antigen expression

Two color immunofluorescence analysis of lymphocyte cell surface antigen expression using an unconjugated plus a biotinylated monoclonal antibody (mAb) requires four incubation steps: (1) unconjugated mAb; (2) fluorochrome-labelled goat anti-mouse Ig; (3) biotinylated mAb; (4) fluorochrome-labelled avidin or streptavidin. We describe a time-saving modification of this procedure which requires only two incubation steps: (1) simultaneous unconjugated and biotinylated mAbs; (2) fluorochrome-labelled avidin/streptavidin followed by fluorochrome-labelled goat anti-mouse Ig. The slightly delayed (5 min) addition of the goat anti-mouse Ig prevents it from binding to the mAb which has already interacted with avidin/streptavidin. Both procedures yield identical results with a variety of different mAbs.