Immunofluorescence staining

For staining of cross sections, eyes of mice and rats were embedded in Tissue-Tek Optimal Cutting Temperature compound (Sakura Seiki, Tokyo, Japan) and then cut into 8-μm sections along the direction of the optical nerve. Sections and HCE monolayer on the slides were fixed with 4% formaldehyde for 20 min and then blocked with 2% donkey serum albumin and 0.1% triton. Next, sections and monolayers were incubated with PAX6 antibody (9013101, Biolegend, San Diego, CA, USA) or SUMO1 antibody (4940, Cell Signaling Technology, USA) overnight at 4 °C, followed by CyTM3-conjugated secondary antibody (Jackson ImmunoResearch, PA, USA) for 60 min at room temperature. Counterstaining was performed with Hoechst 33342 (Invitrogen, California, USA). To compare the expression of PAX6 or SUMO1, Image-Pro Plus software (Media Cybernetics) was used to count fluorescence intensity per area of images taken by confocal (Zeiss Confocal LSM 710 microscope) or fluorescence microscope (Olympus BX51). Three random fields were selected for each sample (n = 3/group).

For evaluating the patterns of the corneal endothelium, staining of flat-mounted corneas was used. Eyes of rats were first fixed with 4% formaldehyde overnight. Then corneas were isolated and blocked with 5% donkey serum albumin and 0.1% triton for 1 h. Next, corneas were incubated with ZO-1 antibody (ab221547, Abcam, MA, USA) overnight at 4 °C, also followed by CyTM3-conjugated secondary antibody and counterstaining as above. Last, corneas were mounted under a coverslip, and the endothelium was viewed and photographed. Three random fields were selected for each sample (n = 3/group).