Immunofluorescent Staining

Decidua basalis specimens were collected from patients with first-trimester elective termination without pregnancy complications or term pregnancy with preeclampsia and from gestational age-matched controls under institutional review board (IRB) approval at Mackay Memorial Hospital, Taipei, Taiwan. Preeclampsia was diagnosed as the existence of elevated blood pressure (≥140/90 mm Hg) and proteinuria (>1+ on a urine dip stick) found twice, 6 hours apart after 20 weeks of gestation without signs and symptoms of chorioamnionitis or chronic villitis. Both preeclampsia and gestational age-matched controls (37–40 weeks) were obtained by cesarean delivery without labor. Serial sections (8 μm) of optimum cutting temperature-embedded decidua were incubated with rabbit antihuman CX3CL1 in Tris-buffered saline (1:500; Sigma-Aldrich, St. Louis, Missouri) for 1 hour at 25°C. Immunostaining specificity was confirmed by substituting a rabbit IgG isotype (DAKO, Santa Clara, California). The sections were then treated with fluorescein isothiocyanate–conjugated donkey antirabbit antibody for 1 hour. After washing, the sections were incubated with goat antihuman vimentin or mouse antihuman cytokeratin 7 (CK-7) antibody for 1 hour at 25°C followed by incubation with rhodamine-conjugated rabbit antigoat antibody (1:400; Sigma-Aldrich) or donkey antimouse antibody (1:500; Millipore; Temecula, California) for 1 hour. Finally, the sections were stained with 4′,6′-diamidino-2-phenylindole (DAPI; Sigma-Aldrich) and mounted in a nonfade mounting medium (DAKO). Immunofluorescence was examined under a Zeiss microscope (Müchen-Hallbergmoos, Germany) equipped with a cooled charge-coupled device camera (Axiocam HRm, Zeiss).