Methanol

Corneas were cut and fixed in 10% neutral buffered formalin (Beijing Yili Fine Chemicals Co., Ltd.; Beijing, China) for 24 h. Paraffin-embedded tissue sections (4 μm) were deparaffinized, rehydrated, and treated with 0.3% hydrogen peroxide in methanol (Beyotime Institute of Biotechnology, Inc., Haimen, China) for 30 min, to eliminate endogenous peroxidase activity. The tissue sections were incubated for 60 min at room temperature with rabbit anti-mouse CXCR4 polyclonal antibody (1:200 dilution; cat. no. ab2074; Abcam). Following three washes of 3 min with phosphate-buffered saline (PBS; Beyotime Institute of Biotechnology, Inc.), a 3,3′-diaminobenzidine detection kit (PV9000; ZSGB-BIO, Beijing, China) was used for CXCR4 staining, according to the manufacturer's protocols. Images were captured with the Leica DM4000B biological microscope equipped with a Leica DFC 550 digital camera and Leica Application Suite version 4.2.0 software (Leica Microsystems GmbH, Wetzlar, Germany).

The cellular migration assay was assessed using Transwell chambers with a pore size of 8 µm (Corning Incorporated, Cambridge, MA, USA). Following transfection for 48 h, 5×10⁴ transfected cells (miR-592 and NC) in 300 µl medium without FBS were seeded into the upper chamber of the Transwell, while 500 µl medium supplemented with 20% FBS was placed into the lower chamber. For the Matrigel invasion assay, the Transwell chamber was coated with Matrigel (BD Biosciences, San Jose, CA, USA). A total of 5×10⁴ transfected cells (miR-592 and NC) in 300 µl medium without FBS were seeded into the upper chamber of the Transwell, while 500 µl medium supplemented with 20% FBS was placed into the lower chamber. Cells were incubated at 37°C for a further 24 h for the migration assay and 48 h for the invasion assay. In the two assays, the cells were fixed with 100% methanol for 5 min (Beyotime Institute of Biotechnology, Haimen, China) and stained with 0.5% crystal violet (Beyotime Institute of Biotechnology) for 5 min. Following this, cells remaining on the upper surface of the membranes were removed carefully using cotton swabs. The migrated and invaded cells were then counted in five randomly selected fields with an inverted microscope (Olympus Corporation, Tokyo, Japan). Each experiment was repeated ≥times.

Transwell inserts (Costar; Corning, Inc.) precoated with Matrigel (BD Biosciences) were used to examine cell invasion. In total, ~10⁵ transfected HTR-8/SVneo cells were inoculated in the upper well in 250 µl RPMI 1640 medium, whereas the lower well contained 500 µl RPMI 1640 medium supplemented with 10% FBS (Sigma-Aldrich; Merck KGaA). After a 24-h incubation at 37°C with 5% CO₂, non-invasive cells attached to the upper side of membranes were removed using cotton swabs. The invasive cells were fixed with 100% methanol (Beyotime Institute of Biotechnology) for 20 min at 37°C and stained with 0.1% crystal violet (Beyotime Institute of Biotechnology) for 10 min at 37°C. Stained cells were photographed and counted in five randomly-selected fields of view using a light microscope (DMI6000 B microscope; Leica Microsystems GmbH; magnification, ×200).