Double staining technique using a combination of indirect and direct immunofluorescence with monoclonal antibodies

A simple method for double staining by immunofluorescence is described. If for double staining using monoclonal antibodies of the same species only one antibody is conjugated with FITC or TRITC, a combination of indirect and direct immunofluorescence is possible. For cell staining the following incubation steps are carried out: Monoclonal antibody I (unlabelled, mouse), anti-mouse immunoglobulin serum FITC- or TRITC-conjugated, normal mouse serum for blocking of free binding sites of the anti-mouse immunoglobulin, and monoclonal antibody II (mouse) which is conjugated with an alternative fluorochrome. The use of this method is demonstrated for investigation of single cell suspensions (performed as a slide test) and of cryostat sections.