Identification of lymphohemopoietic cells in the kidneys of normal rats

Recently developed monoclonal antibodies against rat lymphohematopoietic cells provide ideal probes for study of the role of the cellular immune system in experimental renal disease. Techniques for optimal and reliable labeling of cells present within the glomeruli have not yet been established. In this study it is shown that a small number of lymphoid and mononuclear cells can be identified within normal rat glomeruli present on frozen kidney sections (4 mu) when indirectly stained with monoclonal antibody W3/13, W3/25, OX1, OX3, or OX8 with the use of sequential incubations with F(ab')2 fragments of 2 fluorochrome-labeled antibodies, the nuclear stain ethidium bromide, and p-phenylenediamine added to retard fluorescence quenching. Cell counts showed good correlation with those obtained with the use of intact glomeruli isolated simultaneously from the same kidneys (r = 0.95 for saline-perfused kidneys; r = 0.99 for exsanguinated kidneys). Studies using isolated glomeruli pretreated with trypsin and DNAase failed to provide any advantage, because the enzymes did not enhance cellular reactivity with W3/13, OX8, or OX3, whereas the W3/25-reactive epitope was completely destroyed. It was only the OX1-reactive epitope which was enhanced by enzyme pretreatment. Thus, the described technique can accurately quantitate glomerular lymphohemopoietic cells on sections of frozen kidney and should provide a reliable method for the study of renal disease.