The eyes were processed for paraffin sectioning. For the anti-α3 (IV) antibody, epitope retrieval was performed by heating slides in 0.2-M HCl, pH = 0.9, in the 2100 Antigen Retriever (Aptum Biologics, Southampton, UK), followed by soaking in 0.1-M glycine/6-M urea solution, pH = 3.2, for 30 minutes at room temperature. For all other antibodies, epitope retrieval was performed using 1× Citrate Plus buffer, pH = 6, at 56°C overnight. Sections were blocked in 3% goat serum in staining buffer for 2 hours at room temperature. Primary antibodies—anti-α3 (IV) (1:200, 7076; Chondrex, Redmond, WA, USA), anti-α4 (IV) (1:200, 7073; Chondrex), anti-transforming growth factor-β1 (TGF-β1; 1:200, 21898-1-AP; Proteintech, Rosemont, IL, USA), anti-TGF-β2 (1:200, 19999-1-AP; Proteintech), anti-connective tissue growth factor (CTGF; 1:100, ab6992; Abcam, Cambridge, UK), and anti-β-catenin (1:100, 9582; Cell Signaling Technology, Danvers, MA, USA)—were incubated overnight at 4°C. The secondary antibody—Alexa Fluor 555 Goat Anti-Rat (A-21434; Thermo Fisher Scientific) and Alexa Fluor 555 Goat Anti-Rabbit (1:800, A-21428; Thermo Fisher Scientific)—was incubated for 2 hours. VECTASHIELD Antifade Mounting Medium with DAPI (VectorLabs, Burlingame, CA, USA) was added, and a coverslip was placed and sealed with nail varnish.
For picrosirius red staining, sections were soaked in picrosirius red solution (0.5 g Sirius red F3B mixed with 500 mL of saturated aqueous picric acid solution) for 60 minutes, then quickly dipped in two changes of 0.5% acidified water. Slides were then serially dehydrated and mounted with resinous medium. Photomicrographs of the retina, cornea, and lens were taken under a confocal microscope (Zeiss LSM 510 META; ZEISS Microscopy, Jena, Germany).