The whole eyeball together with the eyelids and conjunctiva was embedded in Tissue-Tek® O.C.T.™ compound (Sakura Finetek Germany GmbH, Staufen, Germany) and snap frozen in liquid nitrogen. Actively proliferating cells in the corneal epithelium were identified by immunofluorescence on central sagittal eye globe cryosections (10 μm) using an antibody directed against the Ki-67 protein, a cell proliferation marker expressed during active phases of the cell cycle [49]. Tissue sections were fixed in 4% paraformaldehyde for 20 min and washed in phosphate-buffered saline (PBS) twice for 5 min. Subsequently, the tissue sections were blocked with 1% bovine serum albumin for 30 min. The primary antibody (Ki-67 monoclonal antibody from rabbit, MA5-14520, Thermo Fisher Scientific GmbH, Dreieich, Germany) was diluted (1:300 in PBS), placed on the sections and incubated overnight at 4 °C. Next, the slices were washed three times in PBS for 5 min and the secondary antibody (goat anti-rabbit IgG H&L, TRITC-conjugated, catalog number: ab6718, Abcam, Cambridge, UK), diluted 1:200 in PBS, was applied for 1 h in darkness. Subsequently, slides were washed in PBS again and were mounted using a mounting medium containing 4′6-diamidino-2-phenylindole (DAPI) (Vectashield Mounting Medium with DAPI, catalog number: H-1200, BIOZOL Diagnostica Vertrieb GmbH, Eching, Germany) to stain the cell nuclei. Tissue sections were viewed and photographed under a fluorescent microscope (Nikon Eclipse E800) equipped with a digital SPOT camera (Nikon Inc., Tokyo, Japan). DAPI- and Ki-67-labelled cells were counted through the entire thickness of the corneal epithelium in a central zone of 880 µm width by a masked evaluator (A.M.) as previously described [50]. The rate of proliferating cells was defined as the percentage of Ki-67-positive cells of all DAPI-positive cells. To determine the number of goblet cells in the conjunctiva, cryosections of 10 µm thickness were cut and stained with periodic acid-Schiff (PAS) reagent. Conjunctival goblet cell density was measured by a blinded evaluator (S.J.) over 100 µm lengths on the palpebral conjunctiva and results were expressed as the number of positive cells per 100 μm.

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