LOCALIZATION OF SECRETORY, MEMBRANE-ASSOCIATED AND CYTOSKELETAL PROTEINS IN RAT TESTIS USING AN IMPROVED IMMUNOCYTOCHEMICAL PROTOCOL THAT EMPLOYS POLYESTER WAX

Immunocytochemistry is a compromise between maintaining. antigenicity and preserving tissue morphology. In the testis, successful immunostai ning results at the level of resolution provided by the light microsco pe have been obtained through use of either frozen or paraffin section s, although both techniques are fraught with limitations. With freezin g, tissue preservation is not optimum, whereas with paraffin embedding , antigenicity is often destroyed. These limitations are not trivial a nd have led to numerous ambiguous results in the literature. In the pr esent study we wish to report the results of immunocytochemical locali zation of various proteins in testis fixed by perfusion with Bouin's f luid and embedded in polyester wax, a ribboning embedding medium for h istology. The advantages of this medium are that it does not require c learing of tissues in xylene solvents before embedding and that unlike paraffin, it liquifies at 38-degrees-C. Because of these two properti es, the polyester was appears to adequately maintain antigenicity as c ompared to that observed in frozen sections, yet because it is a ribbo ning wax, it preserves detailed structure as well as paraffin does. Pr oteins that were immunolocalized included cytoskeletal proteins (tubul in, actin, vinculin, vimentin) and cell-specific markers: 1) androgen- binding protein (ABP) for Sertoli cells; 2) peripheral type benzodiaze pine receptor (PBR) for Leydig cells; and 3) nuclear lamins for germ c ells. Biotin-streptavidin peroxidase immunocytochemistry was employed to determine the specific distribution of the various proteins, and bo th rabbit antisera and mouse monoclonal antibodies were used with equa l success. In addition, fluorochrome-labeled second antibodies combine d with confocal microscopy were used to examine the disposition of the antigens in the testis. Results revealed maintenance of antigenicity and morphology far superior to that obtained with paraffin and frozen sections, respectively, they also showed that within the seminiferous epithelium, germ cell or Sertoli cell-specific proteins were unambiguo usly immunolocalized to their respective cells. Specific observations made possible through use of this protocol suggest that neither tubuli n or vimentin immunostaining patterns in Sertoli cells are altered dur ing the cycle of the seminiferous epithelium. Similarly, ABP staining appeared constant throughout the cycle. Further, we wish to report tha t anti-PBR is a specific probe for Leydig cells in vivo and that an an ti-nuclear lamin antibody appears to serve as a specific probe for spe rmatogonia and pachytene spermatocytes, but that the commercially avai lable anti-smooth muscle alpha-actin monoclonal antibody immunostains both the myoid and lymphatic endothelial cells forming the peritubular cells layer of the seminiferous tubule. We would like to suggest the use of this embedding protocol as a means for immunocytochemical exami nation of protein antigens in the testis.