Immunoenzymatic staining methods for simultaneous demonstration of chromosomes and cell surface markers.

We have developed immunocytochemical staining methods for the simultaneous phenotypic and karyotypic characterization of individual cells. Following a mild hypotonic pretreatment, isolated cells are cytocentrifuged on poly-L-lysine coated slides, fixed in formol buffered acetone, and subsequently labeled with monoclonal antibodies utilizing indirect immunoenzymatic staining procedures with horseradish peroxidase (HRP) or alkaline phosphatase monoclonal anti-alkaline phosphatase (APAAP) as second antibodies. Preparations are refixed consecutively in methanol and 45% acetic acid and counterstained with either "Stains-all" (HRP labeled preparations) or Giemsa (APAAP labeled preparations). C-banding or weak G-banding, which allows the identification of individual chromosomes, can be induced in labeled as well as unlabeled mitotic cells by Ba(OH)2 and/or 2 X SSC treatment after refixation, respectively. Our method has been successfully tested with a variety of monoclonal antibodies against lymphoid, myeloid, erythropoietic, and thrombopoietic cell surface antigens. It is fast, allows the adjustment of the intensity of cell surface staining, and results in permanent preparations suitable for light microscopic analysis.