Immunoenzymatic methods for the study of surface bound immunoglobulins in mice

Abstract

Methods are described for the study of lymphocyte surface bound immunoglobulins (Ig) for light microscopy experimentation. a) Live peripheral lymph node cells (LNC) or spleen cells were reacted in suspension under capping conditions with rabbit anti-mouse Ig antibodies. After washing the cells were cytocentrifuged, fixed and relabeled with either peroxidase-(PO) of fluorescein-(Fl) conjugated anti-rabbit IgG antibodies (Method SC). The staining was heterogeneous and three main categories of lymphocytes were distinguished on the basis of a characteristic surface label distribution (1) all label within distinct caps (caps), (2) all label evenly distributed over the cell surface (rings), and (3) an intermediate pattern where only part of the label was concentrated over one pole (polarized). b) LNC or spleen cells were treated for the simultaneous detection of lymphocyte surface Ig and cytoplasmic Ig of plasma cells. For this, live cells were cytocentrifuged, and after fixation were labeled with PO-conjugated antibodies. Lymphocytes stained in a continuous ring and were easily distinguished on morphological grounds from the intense cytoplasmic stain of plasma cells (Method D). Positive lymphocyte counts done by this method compared favorably with those obtained by Method SC. Comparison of Fl- and PO-conjugated reagents were done by either Method SC or D, by scoring total positive cells. Although either reagent gave closely similar percentage values, the lymphocytes stained by the immunoenzymatic technique contrasted sharply from negative cells and were as a result more easily counted. Other practical advantages of immunoenzymatic staining over immunofluorescence are (1) the simultaneous visualization of cell morphology and type of label distribution over the cell surface, (2) cell scoring can be done rapidly on a large population sample, and (3) stained slides can be reexamined on subsequent days. A common advantages of Method SC for both immunofluorescence and immoenzymology is that antibody labeled slides can be stored before the final staining step.