Detection of leukemic cell colonies in agar plates by immunostaining for human malignancy-associated nucleolar antigen

We have developed an indirect immunofluorescence technique to stain individual colonies in situ in agar to detect the presence of a human malignancy-associated nucleolar antigen. Bone marrow and peripheral blood cells from leukemia patients who were untreated or suffering relapse were used as a source for colony formation. The agar layer containing the colonies was stabilized by overlayering it with an additional agar layer. Cells in the colonies were fixed with methanol, rehydrated, and incubated for prolonged periods in both the primary and the secondary antibodies. The agar was then transferred onto a glass slide, air dried, and examined using a fluorescence microscope. The method is widely applicable for staining colonies in situ in agar with both polyclonal and monoclonal antibodies.