Sc 8427

Four-micrometre tissue sections from formalin-fixed, paraffin-wax-embedded human endometriosis tissues and endometrial tissues were dewaxed, rehydrated, and subjected to high-temperature antigen retrieval. The tissues were incubated with blocking antibody diluent at room temperature for 2 h, and then incubated overnight at 4 °C with the following primary antibodies: anti-vimentin (mouse, ab8978, dilution 1:1000; Abcam), anti-CXCL8 (rabbit, sc-8427, dilution 1:150; Santa Cruz), anti-C7 (rabbit, ab126786, dilution 1:150; Abcam), anti-S100A10 (rabbit, sc-81153, dilution 1:150; Santa Cruz), anti-C3 (rabbit, ab200999, dilution 1:150; Abcam), and anti-StAR (rabbit, sc-166821, dilution 1:150; Santa Cruz). The slides were then incubated with secondary antibody (HRP polymer, anti-mouse/rabbit IgG, Abcam) at room temperature for 1 h. Nuclei were stained with 4-6-diamidino-2-phenylindole (DAPI, ab104139, Abcam) after all antigens had been labelled and then imaged.

Western immunoblotting was carried out as previously described (Wang et al., 2014). In short, thirty micrograms of protein for each sample were denatured in metal bath for 10 min, separated by SDS‐PAGE, and transferred onto polyvinylidene fluoride (PVDF) membranes (Immobilion‐P, Millipore). Membranes were blocked for 1 h with Tris‐buffered saline with Tween‐20 (TBST) containing 5% nonfat dry milk at room temperature, followed by incubation with indicated primary antibodies at 4°C overnight (PRR, 1:1000 dilution, HPA003156, Sigma‐Aldrich; Santa Cruz; fibronectin (FN), 1:1000 dilution, F3648, Sigma‐Aldrich; Collagen 1 (COL‐1), 1:1000 dilution, sc‐59772, Santa Cruz; α‐smooth muscle actin (α‐SMA), 1:1000 dilution, A5228, Sigma‐Aldrich; E‐cadherin, 1:1000 dilution, SAB4503751, Sigma‐Aldrich; interleukin 8 (IL‐8), 1:1000 dilution, sc‐8427, Santa Cruz; transforming growth factor β1 (TGF‐β1), 1:1000 dilution, ab31013, Abcam; Wnt3A, 1:1000 dilution, 09‐162, EMD Millipore; Active‐β‐Catenin, 1:1000 dilution, 05‐665; EMD Millipore; β‐actin, 1:10000 dilution, AA132, Beyotime Biotech Inc.) for overnight at 4°C. Bound antibodies were visualized by using enhanced chemiluminescence technology. Blots were quantified by densitometry using Fluor Chem FC3 image analyzer (Molecular Devices, USA). β‐actin served as the internal reference.

The levels of IL-8 were determined in 16-HBE supernatants using a commercial ELISA kit (R&D Systems, Inc., MN, USA), according to the manufactures' specifications. The lower detection limit for IL-8 was <5 pg/mL. Furthermore, the levels of IL-8 expression were determined in 16-HBE and N-HBE protein extracts by western blot using a mouse monoclonal anti-IL-8 (dilution 1 : 100, overnight at 4°C) (sc-8427, Santa Cruz Biotechnology, Inc.) and a sheep anti-mouse IgG-HRP (dilution 1 : 1000) (NA931, GE Healthcare UK, Little Chalfont, Buckinghamshire, UK) as primary and secondary antibody, respectively.