Expression of the Erythropoietin Receptor: MATERIALS AND METHODS(5)

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.

This entry was posted in Human Placenta and tagged Erythropoietin Receptor, Human Placenta, Trophoblast Cells.