Anti cytokeratin 7

For in vitro studies, we isolated villous trophoblasts from healthy human term placentas by enzymatic digestion and gravitational separation as previously described by Nikitina et al.^{71 (link)} with minor modifications. Briefly, villi-rich tissues were digested four times with 0.25% trypsin (Sigma, USA) and 300 IU/ml Deoxyribonuclease I (Sigma, USA), and then subjected to Percoll^® (Sigma, USA) density gradient centrifugation to isolate villous CTB. In order to assure the purity of the cells, flow cytometry analysis was performed by using the trophoblast-specific epithelial cell marker cytokeratin 7^{72 (link)} (anti-cytokeratin 7, Dako, Switzerland). Vimentin (anti-vimentin, Sigma, USA), which is only expressed in potentially contaminating cells (e.g. mesenchymal cells, fibroblasts, smooth muscle cells, stromal cells)^{73 (link)} served as a negative marker.

Placental villi fixed in 4% paraformaldehyde were included in 5% agarose, sliced in 100 µm sections using a vibratome and permeabilized in 0.5% triton X-100 in PBS for 30 min. Sections were saturated with a solution of 3% bovine serum albumin (BSA) and triton 0.1% X-100 in PBS for 4 h and then incubated overnight at 4 °C under agitation either with the primary antibody, with non-specific rabbit IgG1, or with a mixture of the anti-AhR antibody at 1 µg/mL and the corresponding antigen at 10 µg/mL (APREST78064, Sigma-Aldrich) that correspond to the sequence between 721 and 820 amino acids (C-terminus) of human AhR. Primary antibodies used were: 1 μg/mL anti-AhR antibody #WH0000196-M2, #A9359, #HPA029722, and #HPA029723 (Sigma-Aldrich), #Ab2770 (Abcam, Cambridge, United Kingdom), and 0.75 µg/mL anti-Cytokeratin 7 (#M7018, Dako, Les Ulis, France). After washing, the sections were incubated with Alexa Fluor 555 donkey anti-mouse and Alexa Fluor 488 goat anti-rabbit antibodies in 1% PBS-BSA for 2 h at room temperature. Nuclei were stained with DAPI for 5 min and Vectashield was used as mounting media for confocal microscopy images (Leica TCS SP2 confocal microscope, Leica Microsystems, Nanterre, France).

Villous trophoblast cells were isolated from healthy human term placentas by enzymatic digestion and gravitational separation as previously described (Nikitina et al., 2011 (link); Huang et al., 2013 (link)) with minor modifications. Briefly, villi-rich tissues were digested three times with 0.25% trypsin (Sigma, USA) and 300 IU/ml Deoxyribonuclease I (Sigma, USA), and then subjected to Percoll (Sigma, USA) density gradient centrifugation to isolate villous cytotrophoblast cells. In order to assure the purity of the cells, flow cytometry analysis was performed by using the trophoblast-specific epithelial cell marker cytokeratin 7 (Maldonado-Estrada et al., 2004 (link)) (anti-cytokeratin 7, Dako, Switzerland). Vimentin (anti-vimentin, Sigma, USA), which is only expressed in potentially contaminating cells (e.g. mesenchymal cells, fibroblasts, smooth muscle cells, stromal cells) served as a negative marker (Soares and Hunt, 2006 (link)).