2.3. Histopathology, Immunohistochemistry, and Immunofluorescence

For a histopathological analysis and immunohistochemistry, formalin-fixed, paraffin-embedded tissues were processed as 4 μm thick transverse sections, as previously described [23]. After that, the slides were stained with H&E. Moreover, immunohistochemistry sections were also prepared. For antigen retrieval, the specimens were heated in Target Retrieval Solution (at a low pH, K8005; DAKO, Glostrup, Denmark) with PT-link (DAKO) at 98 °C for 20 min. The slides were then incubated for 10 min at room temperature in hydrogen peroxide to inhibit endogenous peroxidase activity. Before administering the primary antibody, phosphate-buffered saline (PBS) was used to rinse the tissue. The sections were then incubated with anti-AhR (LS-A3391, LS-Bio, Rabbit polyclonal) in a moist chamber at room temperature for one hour. EnVision (Rabbit/Mouse K5007; Dako) was used as the secondary antibody, and 3,3′-diaminobenzidine + chromogen (K5007; Dako) was used to detect positive staining. The sections were counterstained with hematoxylin and then coverslipped. We used the slides incubated with nonimmune rabbit serum instead of the primary antibody as the negative control. As positive controls, the manufacturer-suggested controls were utilized. M.O. and L.M., two independent pathologists, evaluated each histopathological and immunohistochemical staining. As previously described, a semiquantitaive immunohistochemical staining analysis was performed using the H-score for the nuclei and the intensity score for the cytoplasm of the syncytiotrophoblast [10]. In the event of a disagreement, both pathologists examined the specimen and agreed on it.

For an in situ double-marker immunofluorescence analysis of the placenta, two sequential cycles of single-marker immunostaining were performed as previously reported [18,19]. To determine the presence of AhR within the immune cells in addition to the syncytiotrophoblast or cytotrophoblast, fluorescent anti-AhR and anti-CD45 antibodies were used to stain fixed placental sections. CD45 is a transmembrane protein found on all differentiated hematopoietic cells except for erythrocytes and plasma cells [28]. Hofbauer cells, or fetal macrophages, also express CD45 in the placenta [28]. Anti-AhR (LS-A3391, LS-Bio, polyclonal rabbit) and anti-CD45 (Dako, Clone 2B11, monoclonal mouse) were utilized. Following this, A555 and A488 donkey–rabbit fluor-conjugated secondary antibodies (Molec. Probes, Invitrogen) were employed. The slides were examined using a Leica DMI 6000B coupled to a CCD camera (Leica DFC350FX). Two expert pathologists (M.O. and L.M.) blindly evaluated the tissue sections. In the event of a dispute, both pathologists examined the specimen and agreed upon a conclusion. Nuclear co-localization was assessed as previously described, using ImageJ software (v1.53k, http://imagej.nih.gov/ij) [29].