Anti-human MIF and CSF1 goat polyclonal antibodies were purchased from R&D Systems (Abingdon, UK) and Santa Cruz Biotechnology (Santa Cruz, CA), respectively. Horseradish peroxidase-conjugated rabbit anti-goat antibody was obtained from Calbiochem (San Diego, CA). All the chemicals were of analytical grade (Sigma Chemical Co., St. Louis, MO).
Placentas (n = 6) were obtained following cesarean delivery due to term breech presentation. None of the patients were in labor. Approval for this study was granted by the Human Institutional Investigation Committee (HIC) of the University of Siena. Informed consent was obtained from all women. Fullthickness placental blocks were fixed in 10% buffered neutral formalin and embedded in paraffin for histology and immunohistochemistry. For each tissue, a small portion was embedded in OCT (Bio-Optica, Milan, Italy) and frozen in liquid nitrogen. Slides of each specimen were stained with hematoxylin-eosin and examined histologically by a pathologist. Only tissues from uncomplicated pregnancies were included in this study. For each specimen, a block that best represented the maternal decidua was selected for immunohistochemistry.