The reaction was stopped by washing the sections in distilled water, and the slides were mounted and observed under a light microscope. The staining specificity for each cytokine was tested by substituting non-immune serum for the primary antibody.
Isolation and Culture of Decidual Cells
After receiving written informed consent, placentas and attached fetal membranes were obtained from patients with uncomplicated pregnancies who were undergoing repeat cesarean deliveries at term at Yale-New Haven Hospital under HIC approval. None of these patients was in labor. A small portion of each specimen was formalin-fixed, paraffin-embedded, and then examined histologically to rule out underlying inflammation. The decidua was scraped from the maternal surface of the chorion, minced, and digested in Ham F-10 plus 10% charcoal-stripped calf serum (SCS) (Flow Laboratories, Rockville, MD) that contained 25 mg/ml collagenase (200 U/mg) (Worthington Biochemical Corp., Freehold, NJ) in a shaking water bath at 37°C for 30 min. After adding 6.25 U DNase (Sigma) per ml of digestate, the incubation was continued for an additional 45 min.