title: Immunoelectron microscopic identification of human NK cells by FITC-conjugated anti-Leu-11a and biotinylated anti-Leu-7 antibodies.
abstract: Human natural killer (NK) cells have been reported to express various surface antigens. The majority and the most functionally potent NK cells are Leu-11a (NKP-15) positive cells. Only a small number of functional NK cells express Leu-7 (HNK-1) antigen. In the present study, we have established techniques for immunoelectron microscopic identification of NK cells by mouse monoclonal FITC-conjugated anti-Leu-11a and biotinylated anti-Leu-7 antibodies. Ficoll-Hypaque-isolated peripheral blood lymphocytes (PBL) were reacted with the specific antibodies before or after fixation in a 1% glutaraldehyde/1% paraformaldehyde fixative. Prefixation labeling of viable cells with the antibodies was carried out at 4 degrees C or 37 degrees C. Cells prelabeled with anti-Leu-11a antibody were reacted with secondary antibodies either before or after fixation. Anti-Leu-7 antibody was stained directly via an avidin-biotin-peroxidase (ABC) system, anti-Leu-11a antibody was stained indirectly by the ABC immunoperoxidase procedure via a biotinylated anti-mouse IgG secondary antibody or by a 10 nm or 40 nm colloidal gold-labeled anti-mouse IgG antibody. Results indicate that Leu-7 antigen could be localized by incubation with the specific antibody either before or after 20 min fixation; however, Leu-11a antigen was totally abrogated following the same fixation procedure. The Leu-11a antigen was well stained by the methods of prefixation labeling of cells with anti-Leu-11a antibody and incubation with a biotinylated secondary antibody and the ABC system after fixation. With respect to colloidal gold labeling, better results were obtained when cells were reacted with the gold-labeled antibodies immediately after incubation with anti-Leu-11a antibody but before fixation. Ultrastructurally both Leu-7 positive (+) and Leu-11a positive (+) cells shared common ultrastructural features associated with large granular lymphocytes. Using the above described techniques, we found approximately 2-5% Leu-7+ and 9-15% Leu-11a+ cells in the PBL of healthy donors. The overall results suggest that Leu-11a antigen is more sensitive to glutaraldehyde/paraformaldehyde fixation than Leu-7, since it can be localized only by prefixation labeling procedures; the ABC immunoperoxidase procedure is an ideal technique for labeling NK cells for light and electron microscopic enumeration; the immunogold method provides an adequate technique for labeling NK cells which are designated for ultracytochemical studies.
mesh_terms: D000328:Adult; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000954:Antigens, Surface / Q000032:analysis*; D005404:Fixatives / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005557:Formaldehyde / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D006046:Gold; D006801:Humans; D007124:Immunoenzyme Techniques; D007694:Killer Cells, Natural / Q000276:immunology / Q000648:ultrastructure*; D051379:Mice; D008854:Microscopy, Electron; D008875:Middle Aged; D011108:Polymers / Q000494:pharmacology; D013048:Specimen Handling; D013861:Thiocyanates
chemical_list: D000911:Antibodies, Monoclonal; D000954:Antigens, Surface; D005404:Fixatives; D005452:Fluoresceins; D011108:Polymers; D013861:Thiocyanates; D005557:Formaldehyde; D006046:Gold; D016650:Fluorescein-5-isothiocyanate; D005976:Glutaral; C003043:paraform