Ab2770

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).

WI-38 cells were fixed with BD Cytofix/Cytoperm solution for 30 min and then incubated with specific first antibody or isotype control for 30 min at 4°C in the dark. Then the cells were washed and incubated with fluorescent-conjugated second antibody. The following antibodies were used: anti-AhR (ab2770, Abcam) and anti-pSmad2/3(cell signaling). The samples were then analyzed on a FACSCalibur flow cytometer (BD Biosystems).

All the antibodies used in the study were purchased from Abcam: anti-NCOA7 (ab103993, 1/50), anti-Ki67 (ab15580, 1/100), anti-AHR (ab2770, 1/500), anti-CCND1 (ab6152, 1/500), and anti-beta Tublin (ab6046, 1/500).