Fig. 3

The principle of Zenon™ antibody labeling. To label mouse monoclonal IgG antibodies, we employed Zenon™ technology. All steps were performed at room temperature. (a) The unlabeled target monoclonal mouse IgG (purple) is mixed with mouse IgG subclass-specific fluorochrome-conjugated Fab fragments (green/yellow) from the corresponding Zenon™ kit. The fluorochrome-conjugated Fab fragments bind the target antibody. (b) Addition of nonspecific polyclonal mouse immunoglobulin (white) blocks excess unbound Fab fragments. (c) The mix of newly Zenon™-labeled antibodies and blocked excess Fab fragments is added to the cell sample and binds to its respective cell surface antigens. Antibodies (Zenon-labeled or directly conjugated) to other cell surface molecules can be included in this step. (d) Excess antibody and blocked Fab fragments are washed away, and (e) the stained cells are ready to be analyzed or fixed