Simultaneous immunohistochemical demonstration of antigen expression and 5-bromodeoxyuridine incorporation in plastic embedded sections.
In this study a double immunohistochemical staining procedure is described for the simultaneous demonstration of antigen expressing cells and replicating cells in rat thymus. As markers for cell surface antigen expression a monoclonal antibody against Ia-expressing cells (His 19) and a monoclonal antibody against cells of the monocyte-macrophage lineage (ED2) were used. Replicating cells were demonstrated by the incorporation of 5-bromodeoxyuridine (BrdUrd). Tissue pieces were fixed in a periodate-lysine-paraformaldehyde fixative and embedded in glycol methacrylate. To demonstrate Ia-expressing cells or ED2-positive macrophages in plastic embedded sections a digestion with trypsin is necessary. The staining procedure was applied sequentially and was performed with a peroxidase and an alkaline phosphatase labeled reagent yielding respectively a brown and a blue reaction product. Results with this staining procedure on plastic embedded sections of rat thymus, an organ with a high DNA synthesizing capacity, showed incorporation of BrdUrd predominantly in the cortex. ED2-positive macrophages were only found in the cortex. The Ia-positive epithelial reticular cells demonstrated extremely well their stellate form.
D000818:Animals; D000911:Antibodies, Monoclonal; D001973:Bromodeoxyuridine / Q000378:metabolism*; D005260:Female; D000949:Histocompatibility Antigens Class II / Q000032:analysis*; D006652:Histological Techniques; D006735:Horseradish Peroxidase; D007124:Immunoenzyme Techniques; D007137:Immunoglobulin alpha-Chains / Q000032:analysis; D008264:Macrophages / Q000276:immunology; D008297:Male; D010969:Plastics; D051381:Rats; D011919:Rats, Inbred Strains; D013194:Staining and Labeling; D013950:Thymus Gland / Q000166:cytology / Q000276:immunology*
D000911:Antibodies, Monoclonal; D000949:Histocompatibility Antigens Class II; D007137:Immunoglobulin alpha-Chains; D010969:Plastics; D006735:Horseradish Peroxidase; D001973:Bromodeoxyuridine