Western blot analysis
Proteins from tear samples and conjunctival impression cytology were obtained by sonication and centrifugation as previously described.24, 25 Briefly, proteins were transferred onto nitrocellulose membrane by using Trans-blot turbo transfer system (Biorad Laboratories, Milan, Italy), membranes were then blocked with no fat dry milk (5% in PBS-tween) for 1 hour at room temperature, and probed overnight with the following antibodies: anti-TLR9 (sc-25468, Santa Cruz Biotechnology, CA, USA), anti-TLR4 (sc-52962, Santa Cruz Biotechnology, CA, USA), anti-CD14 (sc-58951, Santa Cruz Biotechnology, CA, USA), and anti-actin (sc-47778, Santa Cruz Biotechnology, CA, USA). The day after, following incubation with secondary anti-rabbit and anti-mouse antibodies (Biorad Laboratories, Milan, Italy), protein-bound antibodies were visualized by using chemiluminescence reaction with Clarity Western ECL substrates (Biorad Laboratories, Milan, Italy) and detected through Chemidoc Imaging System (Biorad Laboratories, Milan, Italy). Images derived from Western blots were analyzed with ImageJ software for Windows, and the ratio between the protein of interest and the correspondent housekeeping protein/Ponceau Red staining was calculated. Notably, actin was chosen as reference to normalize protein loading in conjunctival impression cytology, whereas sCD14 from tear samples was normalized by using Ponceau Red staining. Values are expressed as arbitrary units (a.u.).