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.).