Labeling of lymphocyte surface immunoglobulins with T4 as marker for scanning electron microscopy.
The technique originally described by Kumon et al. for the labeling of cell surface antigens under the SEM has been adapted to the study of surface immunoglobulins on human lymphocytes. Briefly, the peripheral blood lymphocytes are: 1) separated in a Ficoll Hypaque gradient, 2) rinsed with culture medium, 3) allowed to attach to plastic coverslips, 4) prefixed with 1% glutaraldehyde for 10 minutes, 5) incubated with goat anti-human immunoglobulins for 30 minutes at 24 degrees C, 6) rinsed, 7) incubated with a rabbit anti-goat IgG coupled with purified bacteriophage T4, 8) rinsed, 9) postfixed in glutaraldehyde for several hours, and 10) dried from ethanol at -75 degrees C and under 10(-2) Torr. Results from normal human lymphocytes and cells from cases of Chronic Lymphoblastic Leukemia (CLL) indicate that the method makes it possible to recognize cells with surface immunoglobulins and permits some correlation between the surface architecture of the cells and the presence of surface Ig. It also permits the study under the SEM of T-derived lymphocytes and of null cells, the unlabeled cell population, without resorting to techniques for lymphocyte subclass separation which may alter surface morphology.
D006801:Humans; D007945:Leukemia, Lymphoid / Q000276:immunology / Q000648:ultrastructure; D008214:Lymphocytes / Q000276:immunology / Q000648:ultrastructure*; D008855:Microscopy, Electron, Scanning / Q000379:methods; D011947:Receptors, Antigen, B-Cell / Q000032:analysis*; D013604:T-Phages / Q000276:immunology*
D011947:Receptors, Antigen, B-Cell