Immunological phenotyping in situ of myeloid colonies in agar cultures

A technique to immunologically stain hematopoietic colonies in agar cultures would make the analysis of clonal aggregates in situ possible without destroying their architecture and avoid the necessity of picking and washing the cells from semi-solid media. A method is described employing mild glutaraldehyde fixation of agar cultures grown directly on polysiloxane-coated slides. This procedure provided agar plugs which were thin enough to allow the complete penetration of reagents without any loss in plating efficiency. Incubation with primary antibodies for 1.5 h and final development in an indirect enzyme immunoassay using an alkaline phosphatase-conjugated secondary antibody gave excellent morphological and immunological results. As an example, the immunological characterization of normal myeloid colonies with a number of monoclonal antibodies is demonstrated.