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Recent developments in the production of monoclonal antibodies have helped considerably in understanding the human immune system and its abnormalities. The distinction of T cell subsets is important not only in studies of the functional characteristics of lymphocytes but also in the investigation of the neoplastic processes of lymphocytes.'-4 Immunoelectron microscopic methods allow identification of lymphocytes by means of their ultrastructural characteristics.56 It is difficult, however, to achieve satisfactory preservation of morphology at the electron microscopic level while preserving antigenicity, and usually all procedures must be carried out successively and without delay after the specimens are taken. We report here a method by which both antigens and ultrastructural morphology can be preserved for at least one year after fixation. Material and methodsTen lymph node biopsies each from T and B cell lymphomas diagnosed by clinical, histological, and immunological examination and five non-neoplastic lymph nodes were used for this study. FIXATION, EMBEDDING, AND STORAGESpecimens were cut into slices less than 1 mm thick. They were fixed in a periodate-lysineparaformaldehyde7 for 4-6 h at 4°C with continuous agitation. Then they were washed in two to three changes of cold phosphate buffered saline (PBS Sections of 10 ,um thickness were cut in a cryostat and mounted on albumin coated slides. The sections on the slides were air dried for at least 1 h and then dried in a desiccator at 4°C overnight to ensure adhesion. They could be preserved in the desiccator at 4°C for three months. Before the sections were used, the desiccator in which they were kept was brought to room temperature in order to prevent condensation on the slides.After washing in two to three changes of cold PBS containing 10% sucrose, the sections were allowed to react with appropriate antibodies-namely, OKT-3, 4 (Ortho Pharmaceuticals Ltd), Leu 3A and Leu 4 (Becton Dickinson Facs Systems), or both; OKT-8, OKIa (Ortho Pharm Ltd); and Bi (Coulter Electronics Inc). For negative control sections normal mouse serum was substituted for each primary antibody.A three step immunoperoxidase technique was applied, using peroxidase-antiperoxidase (PAP) complex (mouse) specifically made for this purpose in our laboratory: first, the sections were allowed to react with the above optimally diluted antibodies in the wet chamber at room temperature for 2 h. Then they were washed in two to three changes of cold PBS containing 10% sucrose for 1 h at 4°C and incubated with biotinylated horse antimouse IgG (Vector Laboratories Inc) for 2 h at room temperature. They were again washed overnight in cold PBS containing 10% sucrose at 4°C and then incubated with both avidin-biotinylated peroxidase complex (Vector Laboratories Inc) and PAP complex (mouse)5 89 for 4 h at room temperature in the wet chamber.After incubation they were washed in cold PBS containing 10% sucrose for 1 h, fixed in 2% glutaraldehyde for 10 min at room temperature, and washed again in two to th...