Preabsorption experiments were conducted with the mh2er 16.5.1 antibody from Genetics Institute. For these studies, the antibody was used at a final concentration of 3 ^g/ml after incubation overnight at 4°C with 3-, 10-, or 30-fold molar excess of soluble EPO-R (Genetics Institute) or of an irrelevant receptor, soluble tumor necrosis factor receptor 1 (TNF-R1) (R & D Systems, Minneapolis, MN). Because of the potential for nonspecific interaction of monoclonal antibodies with cytoskeletal proteins such as cytokeratin, which are abundant in the placenta, the EPO-R antibody was also incubated overnight at 4°C with a 30-fold molar excess of the most abundant cyto-keratin types in the human placenta, cytokeratins 8 and 18 (Cortex Biochem, San Leandro, CA), or an equal volume of vehicle. In parallel experiments, the monoclonal antibody for cytokeratin 18 (clone CY-90; Sigma) was preabsorbed with the cytokeratin 8/18 to confirm the binding potential of the antigen. ampicillin antibiotic
In order to determine whether EPO-R staining was localized to the trophoblast cells, immunocytochemical procedures were performed on adjacent tissue sections using a mouse anti-human cytokeratin monoclonal antibody (1.75 ^g/ml; Sigma) to identify the syncytiotrophoblast layer and underlying cytotrophoblast cells of floating villi, as well as extravillous cytotrophoblast cells in the basal plate.