Anti fibronectin

A single-cell suspension was prepared by filtering the homogenate using a 40-μm pore cell strainer (BD Pharmingen, San Jose, CA, USA). Cells were incubated with Fc Block anti-CD16/32 (IC1918F, BD Pharmingen) and stained with fluorescein isothiocyanate-conjugated anti-fibronectin or isotype control (IC002F, R&D Systems) for 1 h. Fibronectin-positive cells and E-cadherin-positive cells were analyzed using a BD FACS Diva instrument (version 8.0; BD Biosciences). Fibrosis was induced in hTECs with 2 ng/mL rTGF-β (R&D Systems) for 48 h. To evaluate the role of CCL8 in fibrosis, rTGF-β-stimulated hTECs were simultaneously treated with an anti-CCL8 monoclonal antibody (50 or 100 ng/mL; Invitrogen). To quantify cell fibrosis and adhesion, cells were harvested and stained with antibodies against fibronectin (Invitrogen) and E-cadherin (R&D Systems) according to the manufacturers’ protocols. Apoptosis and necrosis were quantified by flow cytometry using an annexin V/propidium iodide assay. hTECs stained with propidium iodide and FITC-conjugated annexin V were incubated for 30 min in the dark, followed by analysis with the BD FACS Diva instrument.

The primary antibodies used were: anti-filaggrin (1:500; Abcam, catalog number # ab24584), anti-fibronectin (1:200; R&D Systems, catalog number # AF19181), anti-GFAP (1:400; R&D Systems, catalog number # AF2594), anti-βIII tubulin (1:200; R&D Systems, catalog number # MAB1195), anti-vimentin (1:200; R&D Systems, catalog number # MAB2105), anti-nestin (1:200; R&D Systems, catalog number # MAB2736), anti-CNPase (1:400; Chemicon, catalog number # MAB326), CD34-FITC (Pharmingen, San Jose, CA catalog number # 553733) and anti-p63 (1:500, AbCAM, catalog number # ab53039). The secondary antibodies were: Texas Red anti-sheep (1:1,000 catalog number # TI-6000), FITC anti-sheep (1:500 catalog number # F7634), FITC anti-rabbit (1:1,000 catalog number # FI-1000), anti-sheep HRP (1:1,000 catalog number # A16-147), anti-rabbit HRP (1:1,000 catalog number # PI-1000) all from Vector Laboratories, USA. The fluorophore-conjugated antibodies for flow cytometry were: CD34-PE catalog number # 551387, CD31-APC catalog number #561814, CD45-PE catalog number # 553081, CD44-PE catalog number # 561860, CD90-FITC catalog number # 554894, CD29-PE catalog number #562801, CD105-PE catalog number # 562759 diluted 1:500, all from BD Biosciences (San Jose, CA, USA).